Bioeffective krill oil compositions

ABSTRACT

This invention discloses new krill oil compositions characterized by having high amounts of phospholipids, astaxanthin esters and/or omega-3 contents. The krill oils are obtained from krill meal using supercritical fluid extraction in a two stage process. Stage 1 removes the neutral lipid by extracting with neat supercritical CO 2  or CO 2  plus approximately 5% of a co-solvent. Stage 2 extracts the actual krill oils by using supercritical CO 2  in combination with approximately 20% ethanol. The krill oil materials obtained are compared with commercially available krill oil and found to be more bioeffective in a number of areas such as anti-inflammation, anti-oxidant effects, improving insulin resistances and improving blood lipid profile.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of pending U.S. patent applicationSer. No. 12/057,775, filed Mar. 28, 2008, which claims the benefit ofexpired U.S. Provisional Patent Application No. 60/920,483, filed Mar.28, 2007, expired U.S. Provisional Patent Application No. 60/975,058,filed Sep. 25, 2007, expired U.S. Provisional Patent Application No.60/983,446, filed Oct. 29, 2007, and expired U.S. Provisional PatentApplication No. 61/024,072, filed Jan. 28, 2008, all of which areincorporated by reference herein in their entirety.

FIELD OF THE INVENTION

This invention relates to extracts from Antarctic krill that comprisebioactive fatty acids.

BACKGROUND OF THE INVENTION

In the Southern Ocean, off the coast of Antarctica, Antarctic krill(Euphausia superba) can be found in large quantities, ranging from300-500 million metric tons of biomass. It feeds on phytoplankton duringthe short Antarctic summer. During winter, however, its food supply islimited to ice algae, bacteria, marine detritus as well as depletingbody protein for energy.

In order to isolate the krill oil from the krill, solvent extractionmethods have been used. See, e.g., WO 00/23546. Krill lipids have beenextracted by placing the material in a ketone solvent (e.g. acetone) inorder to extract the lipid soluble fraction. This method involvesseparating the liquid and solid contents and recovering a lipid richfraction from the liquid fraction by evaporation. Further processingsteps include extracting and recovering by evaporation the remainingsoluble lipid fraction from the solid contents by using a solvent suchas ethanol. See, e.g., WO 00/23546. The compositions produced by thesemethods are characterized by containing at least 75 μg/g astaxanthin,preferably 90 μg/g astaxanthin. Another krill lipid extract disclosedcontained at least 250 μg/g canastaxanthin, preferably 270 μg/gcanastaxanthin.

Krill oil compositions have been described as being effective fordecreasing cholesterol, inhibiting platelet adhesion, inhibiting arteryplaque formation, preventing hypertension, controlling arthritissymptoms, preventing skin cancer, enhancing transdermal transport,reducing the symptoms of premenstrual symptoms or controlling bloodglucose levels in a patient. See, e.g., WO 02/102394. In yet anotherapplication, a krill oil composition has been disclosed comprising aphospholipid and/or a flavonoid. The phospholipid content in the krilllipid extract could be as high as 60% w/w and the EPA/DHA content ashigh as 35% (w/w). See, e.g., WO 03/011873.

Furthermore, nutraceuticals, pharmaceuticals and cosmetics comprisingthe phospholipid extract were disclosed. Previously, it was also shownthat supercritical fluid extraction using neat CO₂ could be used toprevent the extraction of phospholipids in order to extract the neutrallipid fraction from krill, which comprised of esterified and freeastaxanthin. See, e.g., Yamaguchi et al., J. Agric. Food Chem. (1986),34(5), 904-7. Supercritical fluid extraction with solvent modifier haspreviously been used to extract marine phospholipids from salmon roe,but has not been previously used to extract phospholipids from krillmeal. See, e.g., Tanaka et al., J. Oleo Sci. (2004), 53(9), 417-424.

The methods described above rely on the processing of frozen krill thatare transported from the Southern Ocean to the processing site. Thistransportation is both expensive and can result in degradation of thekrill starting material. Data in the literature showing a rapiddecomposition of the oil in krill explains why some krill oil currentlyoffered as an omega-3 supplement in the marketplace contains very highamounts of partly decomposed phosphatidylcholine and also partlydecomposed glycerides. Saether et al., Comp. Biochem Phys. B 83B(1):51-55 (1986). The products offered also contain high levels of freefatty acids.

What is needed in the art are methods for processing krill that do notrequire transport of frozen krill material over long distances and theproducts produced by those methods.

SUMMARY OF THE INVENTION

In a first aspect of the invention is a composition characterized bycomprising at least 65% (w/w) phospholipids.

In another aspect of the invention is a composition obtained fromaquatic or marine sources, characterized by comprising 65% (w/w)phospholipids.

In yet another aspect of the invention is a composition obtained fromkrill, characterized by comprising at least 65% (w/w) phospholipids.

In another aspect of the invention is a composition obtained from krill,characterized by comprising at least 65% (w/w) phospholipids and atleast 39% omega-3 fatty acids (w/w).

In yet another aspect of the invention is a composition obtained fromkrill, characterized by comprising at least 65% (w/w) phospholipids, atleast 39% omega-3 fatty acids (w/w) and at least 580 mg/kg astaxanthinesters.

In another aspect of the invention is a composition obtained from krill,characterized by comprising at least 39% omega-3 fatty acids (w/w) andat least 580 mg/kg astaxanthin esters.

In yet another aspect of the invention is a composition obtained fromkrill, characterized by comprising at least 65% (w/w) phospholipids andat least 580 mg/kg astaxanthin esters.

In yet another aspect, the present invention provides a krill oileffective for reducing insulin resistance, improving blood lipidprofile, reducing inflammation or reducing oxidative stress.

In some embodiments, the present invention provides compositionscomprising: from about 3% to 10% ether phospholipids on a w/w basis;from about 35% to 50% non-ether phospholipids on w/w basis, so that thetotal amount of ether phospholipids and non-ether phospholipids in thecomposition is from about 48% to 60% on a w/w basis; from about 20% to45% triglycerides on a w/w basis; and from about 400 to about 2500 mg/kgastaxanthin. In some embodiments, the ether phospholipids are selectedfrom the group consisting of alkylacylphosphatidylcholine,lyso-alkylacylphosphatidylcholine, alkylacylphosphatidylethanolamine,and combinations thereof. In some embodiments, the ether lipids aregreater than 90% alkylacylphosphatidylcholine. In some embodiments, thenon-ether phospholipids are selected from the group consisting ofphosphatidylcholine, phosphatidylserine, phosphatidylethanolamine andcombinations thereof. In some embodiments, krill oil compositioncomprises a blend of lipid fractions obtained from krill. In somepreferred embodiments, krill is Euphausia superba, although other krillspecies also find use in the present invention. Other krill speciesinclude, but are not limited to E. pacifica, E. frigida, E.longirostris, E. triacantha, E. vallentini, Meganyctiphanes norvegica,Thysanoessa raschii and Thysanoessa inermis. In some embodiments, thecompositions comprise from about 25% to 30% omega-3 fatty acids as apercentage of total fatty acids and wherein from about 80% to 90% ofsaid omega-3 fatty acids are attached to said phospholipids. In someembodiments, the present invention provides a capsule containing theforegoing compositions.

In further embodiments, the present inventions provide compositionscomprising: from about 3% to 10% ether phospholipids on a w/w basis; andfrom about 400 to about 2500 mg/kg astaxanthin. In some embodiments, thecompositions further comprise from about 35% to 50% non-etherphospholipids on w/w basis, so that the total amount of etherphospholipids and non-ether phospholipids in the composition is fromabout 38% to 60% on a w/w basis. In some embodiments, the compositionsfurther comprise from about 20% to 45% triglycerides on a w/w basis. Insome embodiments, the ether phospholipids are selected from the groupconsisting of alkylacylphosphatidylcholine,lyso-alkylacylphosphatidylcholine, alkylacylphosphatidylethanolamine,and combinations thereof. In some embodiments, the ether lipids aregreater than 90% alkylacylphosphatidylcholine. In some embodiments, thenon-ether phospholipids are selected from the group consisting ofphosphatidylcholine, phosphatidylserine, phosphatidylethanolamine andcombinations thereof. In some embodiments, krill oil compositioncomprises a blend of lipid fractions obtained from krill. In somepreferred embodiments, krill is Euphausia superba, although other krillspecies also find use in the present invention. Other krill speciesinclude, but are not limited to E. pacifica, E. frigida, E.longirostris, E. triacantha, E. vallentini, Meganyctiphanes norvegica,Thysanoessa raschii and Thysanoessa inermis. In some embodiments, thecompositions comprise about 25% to 30% omega-3 fatty acids as apercentage of total fatty acids and wherein from about 80% to 90% ofsaid omega-3 fatty acids are attached to said phospholipids. In someembodiments, the present invention provides a capsule containing theforegoing compositions.

In some embodiments, the present invention provides a compositioncomprising at least 65% (w/w) of phospholipids, said phospholipidscharacterized in containing at least 35% omega-3 fatty acid residues. Insome preferred embodiments, the composition is derived from a marine oraquatic biomass. In some further preferred embodiments, the compositionis derived from krill. In some embodiments, the composition comprisesless than 2% free fatty acids. In some embodiments, compositioncomprises less than 10% triglycerides. In some preferred embodiments,the phospholipids comprise greater than 50% phosphatidylcholine. In someembodiments, the composition comprises at least 500 mg/kg astaxanthinesters. In some embodiments, the composition comprises at least 500mg/kg astaxanthin esters and at least 36% (w/w) omega-3 fatty acids. Insome embodiments, the composition comprises less than about 0.5 g/100 gtotal cholesterol. In some embodiments, the composition comprises lessthan about 0.45% arachidonic acid (w/w).

In some embodiments, the present invention provides a krill lipidextract comprising at least 500, 100, 1500, 2000, 2100, or 2200 mg/kgastaxanthin esters and at least 36% (w/w) omega-3 fatty acids. Infurther embodiments, the present invention provides a krill lipidextract comprising at least 100 mg/kg astaxanthin esters, at least 20%(w/w) omega-3 fatty acids, and less than about 0.45% arachidonic acid(w/w).

In some embodiments, the present invention provides methods comprisingadministering the foregoing compositions to a subject in an amounteffective for reducing insulin resistance, reducing inflammation,improving blood lipid profile and reducing oxidative stress.

In some embodiments, the present invention provides a krill lipidextract comprising greater than about 80% triglycerides and greater thanabout 90, 100, 500, 1000, 1500, 200, 2100 or 2200 mg/kg astaxanthinesters. In some embodiments, the krill lipid extract is characterized incontaining from about 5% to about 15% omega-3 fatty acid residues. Insome embodiments, the krill lipid extract is characterized in containingless than about 5% phospholipids. In some embodiments, the krill lipidextract is characterized in comprising from about 5% to about 10%cholesterol.

In some embodiments, the present invention provides a krill mealcomposition comprising less than about 50 g/kg total fat. In someembodiments, the krill meal composition comprises from about 5 to about20 mg/kg astaxanthin esters. In some embodiments, the krill mealcomposition comprises greater than about 65% protein. In someembodiments, the krill meal composition of comprises greater than about70% protein. In some further embodiments, the present invention providesan animal feed comprising the krill meal composition.

In some embodiments, the present invention provides methods ofincreasing flesh coloration in an aquatic species comprising feedingsaid aquatic species a composition comprising the krill meal describedabove. In some embodiments, the present invention provides methods ofincreasing growth and overall survival rate of aquatic species byfeeding the krill meal described above.

In some embodiments, the present invention provides methods of producingkrill oil comprising: a) providing krill meal; and b) extracting oilfrom said krill meal. In some embodiments, the krill meal is produced byheat-treating krill. In some embodiments, the krill meal is stored priorto the extraction step. In some embodiments, the extracting stepcomprises extraction by supercritical fluid extraction. In someembodiments, the supercritical fluid extraction is a two step processcomprising a first extraction step with carbon dioxide and a lowconcentration of a co-solvent (e.g., from about 1-10% co-solvent) and asecond extraction step with carbon dioxide and a high concentration of aco-solvent (e.g., from about 10-30% co-solvent). In preferredembodiments, the co-solvent is a C₁-C₃ monohydric alcohol, preferablyethanol. In some embodiments, the present invention provides oilproduced by the foregoing method.

In some embodiments, the present invention provides methods ofproduction of krill oil comprising: a) providing fresh krill; b)treating said fresh krill to denature lipases and phospholipases in saidfresh krill to provide a denatured krill product; and c) extracting oilfrom said denatured krill product. In some embodiments, the denaturationstep comprises heating of said fresh krill. In some embodiments, thedenaturation step comprises heating said fresh krill after grinding. Insome embodiments, the methods further comprise storing said denaturedkrill product at room temperature or below between the denaturation stepand the extraction step. In some embodiments, the enzyme denaturationstep is achieved by application of heat. In some embodiments, theextraction step comprises use of supercritical carbon dioxide, with orwithout use of a polar modifier. In some embodiments, the extractionstep comprises use of ethanol. In some embodiments, the extraction stepis comprises ethanol extraction followed by acetone to precipitation ofphospholipids. In some embodiments, the denatured krill product is ameal. In some embodiments, the present invention provides oil producedby the foregoing method.

In some embodiments, the present invention provides a compositioncomprising oil extracted from krill having a phosphatidylcholine contentof greater then about 50% (w/w). In some embodiments, the oil has aphosphatidylcholine content of greater then about 70% (w/w). In someembodiments, the oil has a phosphatidylcholine content of greater thenabout 80% (w/w). In some embodiments, the composition comprises lessthan 2% free fatty acids. In some embodiments, the composition comprisesless than 10% triglycerides. In some embodiments, the compositioncomprises at least 500 mg/kg astaxanthin esters. In some embodiments,the composition comprises less than about 0.45% arachidonic acid (w/w).

In some embodiments, the present invention provides compositioncomprising odorless krill oil. In some embodiments, the odorless krilloil comprises less than about 10 mg/kg (w/w) trimethylamine. In somefurther embodiments, the present invention provides an odorless krilloil produced by the method comprising: extracting a neutral krill oilfrom a krill oil containing material by supercritical fluid extractionto provide a deodorized krill material, wherein said neutral krill oilcontains odor causing compounds and extracting a polar krill oil fromsaid deodorized krill material by supercritical fluid extraction with apolar entrainer to provide an essentially odorless krill oil.

In some embodiments, the present invention provides a compositioncomprising krill oil containing less than about 70 micrograms/kilogram(w/w) astaxanthin esters. In some embodiments, the compositions compriseless than about 50 micrograms/kilogram (w/w) astaxanthin esters. In someembodiments, the compositions comprise less than about 20micrograms/kilogram (w/w) astaxanthin esters. In some embodiments, thecompositions comprise less than about 5 micrograms/kilogram (w/w)astaxanthin esters.

In some embodiments, the present invention provides a krill oil producedby the process comprising: pumping fresh krill from a trawl onto a ship,heating the krill to provide a krill material, and extracting oil fromthe krill material.

In further embodiments, the present invention provides a blended krilloil composition comprising: from about 45% to 55% w/w phospholipids;from about 20% to 45% w/w triglycerides; and from about 400 to about2500 mg/kg astaxanthin. In some embodiments, the blended krill oilproduct comprises a blend of lipid fractions obtained from Euphausiasuperba. In some embodiments, the composition comprises from about 25%to 30% omega-3 fatty acids as a percentage of total fatty acids andwherein from about 80% to 90% of said omega-3 fatty acids are attachedto said phospholipids.

In still other embodiments, the present invention provides a Euphausiasuperba krill oil composition comprising: from about 30% to 60% w/wphospholipids; from about 20% to 50% triglycerides; from about 400 toabout 2500 mg/kg astaxanthin; and from about 20% to 35% omega-3 fattyacids as a percentage of total fatty acids in said composition, whereinfrom about 70% to 95% of said omega-3 fatty acids are attached to saidphospholipids.

In still further embodiments, the present invention provides a dietarysupplement comprising encapsulated Euphausia superba krill oilcomprising from about 30% to 60% w/w phospholipids; from about 20% to50% triglycerides; from about 400 to about 2500 mg/kg astaxanthin; andfrom about 20% to 35% omega-3 fatty acids as a percentage of total fattyacids in said composition, wherein from about 70% to 95% of said omega-3fatty acids are attached to said phospholipids.

In some embodiments, the present invention provides methods of making aEuphausia superba krill oil composition comprising: contacting Euphausiasuperba with a polar solvent to provide a polar extract comprisingphospholipids; contacting Euphausia superba with a neutral solvent toprovide a neutral extract comprising triglycerides and astaxanthin;combining said polar extract and said neutral extract to provideEuphausia superba krill oil comprising from about 30% to 60% w/wphospholipids; from about 20% to 50% triglycerides; from about 400 toabout 2500 mg/kg astaxanthin; and from about 20% to 35% omega-3 fattyacids as a percentage of total fatty acids in said composition, whereinfrom about 70% to 95% of said omega-3 fatty acids are attached to saidphospholipids. In some embodiments, the methods further comprise thestep of encapsulating the Euphausia superba krill oil. In someembodiments, the present invention provides a Euphausia superba krilloil produced by the methods described above.

In some embodiments, the present invention provides methods of producinga dietary supplement comprising; contacting Euphausia superba with apolar solvent to provide an polar extract comprising phospholipids;contacting Euphausia superba with a neutral solvent to provide a neutralextract comprising triglycerides and astaxanthin; combining said polarextract and said neutral extract to provide Euphausia superba krill oilcomprising from about 30% to 60% w/w phospholipids; from about 20% to50% triglycerides; from about 400 to about 2500 mg/kg astaxanthin; andfrom about 20% to 35% omega-3 fatty acids as a percentage of total fattyacids in said composition, wherein from about 70% to 95% of said omega-3fatty acids are attached to said phospholipids; and encapsulating saidEuphausia superba krill oil.

In some embodiments, the present invention provides methods of reducingdiet-induced hyperinsulinemia, insulin insensitivity, muscle masshypertrophy, serum adiponectin reduction or hepatic steatosis comprisingin a subject exposed to a high fat diet: administering to said subjectexposed to a high fat diet an effective amount of a krill oilcomposition under conditions such that a condition selected from thegroup consisting of diet-induced hyperinsulinemia, insulininsensitivity, muscle mass hypertrophy, serum adiponectin reduction andhepatic steatosis is reduced. The present invention is not limited toany particular krill oil composition. In some embodiments, the krill oilcomposition is a Euphausia superba krill oil composition. The presentinvention is not limited to any particular formulation of krill oil. Insome embodiments, the krill oil composition is encapsulated. In somepreferred embodiments, the effective amount of a krill oil compositionis from 0.2 grams to 10 grams of said krill oil composition. In someembodiments, the krill oil composition comprises: from about 45% to 55%w/w phospholipids; from about 20% to 45% w/w triglycerides; and fromabout 400 to about 2500 mg/kg astaxanthin. In some embodiments, thekrill oil composition comprises a blend of lipid fractions obtained fromEuphausia superba. In some embodiments, the krill oil compositioncomprises from about 25% to 30% omega-3 fatty acids as a percentage oftotal fatty acids and wherein from about 80% to 90% of said omega-3fatty acids are attached to said phospholipids. In some embodiments, thekrill oil composition comprises from about 30% to 60% w/w phospholipids;from about 20% to 50% triglycerides; from about 400 to about 2500 mg/kgastaxanthin; and from about 20% to 35% omega-3 fatty acids as apercentage of total fatty acids in said composition, and wherein fromabout 70% to 95% of said omega-3 fatty acids are attached to saidphospholipids.

In some embodiments, the present invention provides methods of reducingdiet-induced hyperinsulinemia, insulin insensitivity, muscle masshypertrophy, serum adiponectin reduction or hepatic steatosis comprisingin a subject consuming a high fat diet or a normal fat diet:administering to said subject consuming a high fat diet or a normal fatdiet an effective amount of a krill oil composition under conditionssuch that a condition selected from the group consisting of diet-inducedhyperinsulinemia, insulin insensitivity, muscle mass hypertrophy, serumadiponectin reduction and hepatic steatosis is reduced. The presentinvention is not limited to any particular krill oil composition. Insome embodiments, the krill oil composition is a Euphausia superba krilloil composition. The present invention is not limited to any particularformulation of krill oil. In some embodiments, the krill oil compositionis encapsulated. In some preferred embodiments, the effective amount ofa krill oil composition is from 0.2 grams to 10 grams of said krill oilcomposition. In some embodiments, the krill oil composition comprises:from about 45% to 55% w/w phospholipids; from about 20% to 45% w/wtriglycerides; and from about 400 to about 2500 mg/kg astaxanthin. Insome embodiments, the krill oil composition comprises a blend of lipidfractions obtained from Euphausia superba. In some embodiments, thekrill oil composition comprises from about 25% to 30% omega-3 fattyacids as a percentage of total fatty acids and wherein from about 80% to90% of said omega-3 fatty acids are attached to said phospholipids. Insome embodiments, the krill oil composition comprises from about 30% to60% w/w phospholipids; from about 20% to 50% triglycerides; from about400 to about 2500 mg/kg astaxanthin; and from about 20% to 35% omega-3fatty acids as a percentage of total fatty acids in said composition,and wherein from about 70% to 95% of said omega-3 fatty acids areattached to said phospholipids.

In some embodiments, the present invention provides methods of inducingdiuresis in a subject comprising: administering to said subject aneffective amount of a krill oil composition under conditions such thatdiuresis is induced. In some embodiments, the present invention providesmethods of increasing muscle mass in a subject, comprising:

administering to said subject an effective amount of a krill oilcomposition under conditions such that muscle mass is increased. In someembodiments, the present invention provides methods of decreasingprotein catabolism in a subject, comprising: administering to saidsubject an effective amount of a krill oil composition under conditionssuch that protein catabolism is decreased. In some embodiments, thepresent invention provides methods of decreasing lipid content in theheart of a subject, comprising: administering to said subject aneffective amount of a krill oil composition under conditions such thatlipid content in the heart of the subject is decreased. In someembodiments, the present invention provides methods of decreasing lipidcontent in the liver of a subject, comprising: administering to saidsubject an effective amount of a krill oil composition under conditionssuch that lipid content in the liver of the subject is decreased.

DESCRIPTION OF THE FIGURES

FIG. 1. 31P NMR analysis of polar lipids in krill oil.

FIG. 2. Blood lipid profiles in Zucker rats fed different forms ofomega-3 fatty acids (TAG=FO, PL1=NKO and PL2=Superba).

FIG. 3. Plasma glucose concentration in Zucker rats fed different formsof omega-3 fatty acids.

FIG. 4. Plasma insulin concentration in Zucker rats fed different formsof omega-3 fatty acids.

FIG. 5. Estimated HOMA-IR values in Zucker rats fed different forms ofomega-3 fatty acids.

FIG. 6. The effect of dietary omega-3 fatty acids on TNF production byperitoneal macrophages.

FIG. 7. The effect of dietary omega-3 fatty acids on lipid accumulationin the liver.

FIG. 8. The effect of dietary omega-3 fatty acids on lipid accumulationin the muscle.

FIG. 9. The effect of dietary omega-3 fatty acids on lipid accumulationin the heart.

FIG. 10. Relative concentrations of DHA in the brain in Zucker ratssupplemented with omega-3 fatty acids.

FIG. 11. Mean group body weights (g) in the collagen-induced male DBA/1arthritic mice. B—PL2 is the krill oil group. *p<0.05, significantlydifferent from Group A (Positive Control—Fish Oil) and Group C(Control).

FIG. 12. Body weight for the various treatment groups.

FIG. 13. Muscle weight for the various treatment groups.

FIG. 14. Muscle to body weight ratio for the various treatment groups.

FIG. 15. Serum adiopnectin levels (ng/ml) for the various treatmentgroups.

FIG. 16. Serum insulin levels for the various treatment groups.

FIG. 17. Blood glucose (mmol/l) levels in the various treatment groups.

FIG. 18. HOMA-IR values for the various treatment groups.

FIG. 19. Liver triglyceride levels (μmol/g) for the various treatmentgroups.

DEFINITIONS

As used herein, “phospholipid” refers to an organic compound having thefollowing general structure:

wherein R1 is a fatty acid residue, R2 is a fatty acid residue or —OH,and R3 is a —H or nitrogen containing compound choline(HOCH₂CH₂N⁺(CH₃)₃OH⁻), ethanolamine (HOCH₂CH₂NH₂), inositol or serine.R1 and R2 cannot simultaneously be OH. When R3 is an —OH, the compoundis a diacylglycerophosphate, while when R3 is a nitrogen-containingcompound, the compound is a phosphatide such as lecithin, cephalin,phosphatidyl serine or plasmalogen.

An “ether phospholipid” as used herein refers to a phospholipid havingan ether bond at position 1 the glycerol backbone. Examples of etherphospholipids include, but are not limited to,alkylacylphosphatidylcholine (AAPC), lyso-alkylacylphosphatidylcholine(LAAPC), and alkylacylphosphatidylethanolamine (AAPE). A “non-etherphospholipid” is a phospholipid that does not have an ether bond atposition 1 of the glycerol backbone.

As used herein, the term omega-3 fatty acid refers to polyunsaturatedfatty acids that have the final double bond in the hydrocarbon chainbetween the third and fourth carbon atoms from the methyl end of themolecule. Non-limiting examples of omega-3 fatty acids include,5,8,11,14,17-eicosapentaenoic acid (EPA), 4,7,10,13,16,19-docosahexanoicacid (DHA) and 7,10,13,16,19-docosapentanoic acid (DPA).

As used herein, astaxanthin refers to the following chemical structure:

As used herein, astaxanthin esters refer to the fatty acids esterifiedto OH group in the astaxanthin molecule.

As used herein, the term w/w (weight/weight) refers to the amount of agiven substance in a composition on weight basis. For example, acomposition comprising 50% w/w phospholipids means that the mass of thephospholipids is 50% of the total mass of the composition (i.e., 50grams of phospholipids in 100 grams of the composition, such as an oil).

DETAILED DESCRIPTION OF THE INVENTION

This invention discloses novel krill oil compositions characterized bycontaining high levels of astaxanthin, phospholipids, included anenriched quantities of ether phospholipids, and omega-3 fatty acids. Thekrill oils compositions are extracted from krill meal usingsupercritical fluid extraction (SFE) with a co-solvent modifier. Thekrill meal has been processed on board a ship in Antarctica using livekrill as starting material in order to ensure the highest possiblequality of the krill meal. The krill oils are extracted from the krillmeal in two stages, in step 1 the neutral fraction is extracted usingneat supercritical CO₂ or in combination with 5% ethanol. The neutralfraction consisted mostly of triglycerides and cholesterol. In stage 2,the polar lipids (phospholipids) are extracted by adding at least 20%ethanol to the supercritical CO₂ extraction medium.

The present invention provides methods to avoid decomposition ofglycerides and phospholipids in krill oil and compositions produced bythose methods. The product obtained by these new methods is virtuallyfree of enzymatically decomposed oil constituents. The solution to theproblem is to incorporate a protein denaturation step on fresh krillprior to use of any extraction technology. Denaturation can be achievedby thermal stress or by other means. After denaturation, the oil can beextracted by an optional selection of nonpolar and polar solventsincluding use of supercritical carbon dioxide. Krill is adapted to avery efficient nutrient digestion at very low temperatures. Thereforethe enzymes are sensitive to heat and the step of applying thermaldenaturation of lipases and phospholipases does not imply use of veryhigh temperatures. Surprisingly, it has been found that the use of milddenaturation conditions can greatly enhance the quality of krill oil.

Additionally, a major obstacle of several processes of extraction is thecost of removing water. This is particularly true for methods feasiblefor extraction of highly unsaturated lipids where freeze drying has beenregarded as the method of choice to avoid oxidative breakdown of lipids.However, the lipids in krill are surprisingly stable against oxidativedeterioration. Therefore, a process including moderate use of heat inthe water removing process is feasible provided that the enzymes havebeen inactivated.

A. Krill Processing

The present invention provides methods for processing freshly caughtkrill at the site of capture and preferably on board a ship. Afterprocessing on board, the krill can be further subjected to extractionprocesses on board the ship or at a remote location away from the ship.The processing steps described herein also allow for the storage ofkrill material, preferably a krill meal for from about 1, 2, 3, 4, 5, 6,8, 9, 10, 11, or 12 months to about 24 to 36 months prior to processing.

In some preferred embodiments, freshly caught krill is first subjectedto a protein denaturation step. The present invention is not limited toany particular method of protein denaturation. In some embodiments, thedenaturation is accomplished by application of chemicals, heat, orcombinations thereof. In some embodiments, freshly caught krill is wetpressed to obtain oil and meal. In some embodiments, the meal is thenheated to a temperature of about 50° C. to about 100° C. for about 20minutes to about an hour, preferably about 40 minutes to denature theproteins. In some embodiments, this material is then pressed to yield apress cake. When this method is used on krill, only a small amount ofoil is released. Most of the oil is still present in the denatured meal.In some embodiments, antioxidants such as ethoxyquin or Vitamin E areadded to the meal. However, as shown in the examples, the resulting mealis surprisingly stable. The stability can only partly be explained byaddition of an antioxidant to the meal. This antioxidant can, afterextraction of the oil from denatured meal, be removed by furtherprocessing steps. Alternatively the oil can be extracted rather shortlyafter production of the meal without any addition of antioxidant in theprocess. Further, storage conditions at a low to very low temperaturecan be applied if addition of antioxidant is not desired.

Krill oil extracted from denatured krill meal by supercritical fluidextraction even 19 months after the production of the meal containedvirtually no decomposed phospholipids. This product turned out to besubstantially different from samples of krill oil available in themarket today. Previously described commercial krill processingprocedures utilize krill that has been frozen immediately after catchingfollowed by freeze drying and extraction at low temperatures. However,these processes only yield a suitable product if the time the krill iskept frozen is very short or the temperature is extremely low (−60° to−80° C.). However, data provided herein clearly shows that if a step ofdenaturation of the proteins is added in front of an optional extractionmethod, an excellent krill oil can be produced even after a long time ofstorage. This methodology also opens up for use of alternative methodsto remove water prior to extraction, which in turn has a great impact oncosts in full scale operation. If a long time of storage is desired, thedenatured material should preferably be stored at low temperaturepreferably at −20° C.

In some embodiments, krill oil is extracted from the denatured krillmeal. In some embodiments, the krill oil is extracted by contacting thekrill meal with ethanol. In some embodiments, krill is then extractedwith a ketone solvent such as acetone. In other embodiments, the krilloil is extracted by one or two step supercritical fluid extraction. Insome embodiments, the supercritical fluid extraction uses carbon dioxideand neutral krill oil is produced. In some embodiments, thesupercritical fluid extraction uses carbon dioxide with the addition ofa polar entrainer, such as ethanol, to produce a polar krill oil. Insome embodiments, the krill meal is first extracted with carbon dioxidefollowed by carbon dioxide with a polar entrainer, or vice versa. Insome embodiments, the krill meal is first extracted with CO₂supplemented with a low amount of a polar co-solvent (e.g., from about1% to about 10%, preferably about 5%) such a C₁-C₃ monohydric alcohol,preferably ethanol, followed by extraction with CO₂ supplemented with ahigh amount of a polar co-solvent (from about 10% to about 30%,preferably about 23%) such as such a C₁-C₃ monohydric alcohol,preferably ethanol, or vice versa. Surprisingly, it has been found thatuse of a low amount of polar solvent in the CO₂ as an entrainerfacilitates the extraction of neutral lipid components and astaxanthinin a single step. Use of the high of polar solvent as an entrainer inthe other step facilitates extraction of ether phospholipids, as well asnon-ether phospholipids.

The present invention is distinguished from previously described krilloil products, such as those described in U.S. Pat. No. 6,800,299 or WO03/011873 and Neptune brand krill oil, by having substantially higherlevels of non-ether phospholipids, ether phospholipids, and astaxanthin.The krill oils of the present invention also have unexpected andsuperior properties as compared to previously available krill oils. Inparticular, the krill oil of the present invention has been demonstratedto reduce blood LDL cholesterol levels, improve DHA transfer to thebrain as well as reduce lipid accumulation in the liver and muscle whilethe previously described krill oil compositions do not have such aproperties. Accordingly, in some embodiments, the present inventionprovides a krill oil composition, preferably a Euphausia superba krilloil composition, comprising from about 40% to about 60% w/wphospholipids, preferably from about 45% to 55% w/w phospholipids andfrom about 300 mg/kg astaxanthin to about 2500 mg/kg astaxanthin,preferably from about 1000 to about 2200 mg/kg astaxanthin, morepreferably from about 1500 to about 2200 mg/kg astaxanthin. In somepreferred embodiments, the compositions comprise greater than about1000, 1500, 1800, 1900, 2000, or 2100 mg/kg astaxanthin. In somepreferred embodiments, the krill oil compositions of the presentinvention comprise from about 1%, 2%, 3% or 4% to about 8%, 10%, 12% or15% w/w ether phospholipids or greater than about 4%, 5%, 6%, 7%, 8%, 9%or 10% ether phospholipids. In some embodiments the ether phospholipidsare preferably alkylacylphosphatidylcholine,lyso-alkylacylphosphatidylcholine, alkylacylphosphatidyl-ethanolamine orcombinations thereof. In some embodiments, the krill oil compositionscomprise from about 1%, 2%, 3% or 4% to about 8%, 10%, 12% or 15% w/wether phospholipids and from about 30%, 33%, 40%, 42%, 45%, 48%, 50%,52%, 54%, 55% 56%, 58% to about 60% non-ether phospholipids so that thetotal amount of phospholipids (both ether and non-ether phospholipids)ranges from about 40% to about 60%. One of skill in the art willrecognize that the range of 40% to 60% total phospholipids, as well asthe other ranges of ether and non-ether phospholipids, can include othervalues not specifically listed within the range.

In further embodiments, the compositions comprise from about 20% to 45%w/w triglycerides; and from about 400 to about 2500 mg/kg astaxanthin.In some embodiments, the compositions comprise from about 20% to 35%,preferably from about 25% to 35%, omega-3 fatty acids as a percentage oftotal fatty acids in the composition, wherein from about 70% to 95%, orpreferably from about 80% to 90% of the omega-3 fatty acids are attachedto the phospholipids. In some embodiments, the present inventionprovides encapsulated Euphausia superba krill oil compositions. In someembodiments, the present invention provides a method of making aEuphausia superba krill oil composition comprising contacting Euphausiasuperba with a polar solvent to provide an polar extract comprisingphospholipids, contacting Euphausia superba with a neutral solvent toprovide a neutral extract comprising triglycerides and astaxanthin, andcombining said polar extract and said neutral extract to provide theEuphausia superba krill oils described above. In some embodiments,fractions from polar and non-polar extractions are combined to provide afinal product comprising the desired ether phospholipids, non-etherphospholipids, omega-3 moieties and astaxanthin. In other embodiments,the present invention provides methods of making a Euphausia superba (orother krill species) krill oil comprising contacting a Euphausia superbapreparation such as Euphausia superba krill meal under supercriticalconditions with CO₂ containing a low amount of a polar solvent such asethanol to extract neutral lipids and astaxanthin; contacting mealremaining from the first extraction step under supercritical conditionswith CO₂ containing a high amount of a polar solvent such as ethanol toextract a polar lipid fraction containing ether and non-etherphospholipids; and then blending the neutral and polar lipid extracts toprovide the compositions described above.

The krill oil extracted by the methods of the present invention containsfew enzymatic breakdown products. Examples of the krill oil compositionsof the present invention are provided in Tables 9-24. In someembodiments, the present invention provides a polar krill oil comprisingat least 65% (w/w) of phospholipids, wherein the phospholipids arecharacterized in containing at least 35% omega-3 fatty acid residues.The present invention is not limited to the presence of any particularomega-3 fatty acid residues in the krill oil composition. In somepreferred embodiments, the krill oil comprises EPA and DHA residues. Insome embodiments, the krill oil compositions comprise less than about5%, 4%, 3% or preferably 2% free fatty acids on a weight/weight (w/w)basis. In some embodiments, the krill oil compositions comprise lessthan about 25%, 20%, 15%, 10% or 5% triglycerides (w/w). In someembodiments, the krill oil compositions comprise greater than about 30%,40%, 45%, 50%, 55%, 60%, or 65% phosphatidyl choline (w/w). In someembodiments, the krill oil compositions comprise greater than about 100,200, 300, 400, or 500 mg/kg astaxanthin esters and up to about 700 mg/kgastaxanthin esters. In some embodiments, the present invention provideskrill oil compositions comprising at least 500, 1000, 1500, 2000, 2100,or 2200 mg/kg astaxanthin esters and at least 36% (w/w) omega-3 fattyacids. In some embodiments, the krill oil compositions of the presentinvention comprise less than about 1.0 g/100 g, 0.5 g/100 g, 0.2 g/100 gor 0.1 g/100 g total cholesterol. In some embodiments, the krill oilcompositions of the present invention comprise less than about 0.45

In some embodiments, the present invention provides a neutral krill oilextract comprising greater than about 70%, 75% 80%, 85% or 90%triglycerides. In some embodiments, the krill oil compositions comprisefrom about 50 to about 2500 mg/kg astaxanthin esters. In someembodiments, the krill oil compositions comprise from about 50, 100,200, or 500 to about 750, 1000, 1500 or 2500 mg/kg astaxanthin esters.In some embodiments, the compositions comprise from about 1% to about30% omega-3 fatty acid residues, and preferably from about 5%-15%omega-3 fatty acid residues. In some embodiments, the krill oilcompositions comprise less than about 20%, 15%, 10% or 5% phospholipids.

In some embodiments, the present invention provides krill oil containingless than about 70, 60, 50, 40, 30, 20, 10, 5 or 1 micrograms/kilogram(w/w) astaxanthin esters. In some embodiments, the krill oil is clear oronly has a pale red color. In some embodiments, the low-astaxanthinkrill oil is obtained by first extracting a krill material, such askrill oil, by supercritical fluid extraction with neat carbon dioxide.It is contemplated that this step removes astaxanthin from the krillmaterial. In some embodiments, the krill material is then subjected tosupercritical fluid extraction with carbon dioxide and a polar entrainersuch as ethanol, preferably about 20% ethanol. The oil extracted duringthis step is characterized in containing low amounts of astaxanthin. Inother embodiments, krill oil comprising astaxanthin is extracted bycountercurrent supercritical fluid extraction with neat carbon dioxideto provide a low-astaxanthin krill oil.

In some embodiments, the present invention provides krill oil that issubstantially odorless. By substantially odorless it is meant that thekrill oil lacks an appreciable odor as determined by a test panel. Insome embodiments, the substantially odorless krill oil comprises lessthan about 10, 5 or 1 milligrams/kilogram trimethylamine. In somepreferred embodiments, the odorless krill oil is produced by firstsubjecting krill material to supercritical fluid extraction with neatcarbon dioxide to remove odor causing compounds such as trimethylamine,followed by extraction with carbon dioxide with a polar entrainer suchas ethanol.

In some embodiments, the present invention provides a delipidated krillmeal produced after extraction of lipids from the krill meal. In someembodiments, the delipidated krill meal comprises krill protein. In someembodiments, the delipidated krill meal comprises less than about 200,150, 120, 100, 75, 65, 60, 55, or 50 g/kg total fat. In someembodiments, the delipidated krill meal comprises from about 1 to about100 mg/kg astaxanthin esters, and preferably from about 5 to about 20mg/kg astaxanthin esters. In some embodiments, the delipidated krillmeal comprises greater than about 60%, 65%, 70% or 75% krill protein. Insome embodiments, the present invention provides animal feeds comprisingthe delipidated krill meal. In some embodiments, the animal feed is afish feed or aquatic organism feed, such as shrimp feed, crab feed, orcrawfish feed. In preferred embodiments, the krill meal is incorporatedinto complete ration for the target organism. In preferred embodiments,the feed is provided in pelleted form. In many instances, compounds suchas astaxanthin are removed during delipidation. The methods of thepresent invention provide a delipidated krill meal that retainssignificant amounts of astaxanthin. Accordingly, in some embodiments,the present invention provides methods of feeding aquatic organisms,comprising providing to the aquatic organism a feed comprising thedelipidated krill meal described above. In other embodiments, thepresent invention provides methods of increasing flesh coloration in anaquatic species comprising feeding the aquatic species a comprising thedelipidated krill meal described above.

B. Compositions Containing Krill Oil

In some embodiments, the compositions of this invention (such as thosedescribed in the preceding sections) are contained in acceptableexcipients and/or carriers for oral consumption. The actual form of thecarrier, and thus, the composition itself, is not critical. The carriermay be a liquid, gel, gelcap, capsule, powder, solid tablet (coated ornon-coated), tea, or the like. The composition is preferably in the formof a tablet or capsule and most preferably in the form of a soft gelcapsule. Suitable excipient and/or carriers include maltodextrin,calcium carbonate, dicalcium phosphate, tricalcium phosphate,microcrystalline cellulose, dextrose, rice flour, magnesium stearate,stearic acid, croscarmellose sodium, sodium starch glycolate,crospovidone, sucrose, vegetable gums, lactose, methylcellulose,povidone, carboxymethylcellulose, corn starch, and the like (includingmixtures thereof). Preferred carriers include calcium carbonate,magnesium stearate, maltodextrin, and mixtures thereof. The variousingredients and the excipient and/or carrier are mixed and formed intothe desired form using conventional techniques. The tablet or capsule ofthe present invention may be coated with an enteric coating thatdissolves at a pH of about 6.0 to 7.0. A suitable enteric coating thatdissolves in the small intestine but not in the stomach is celluloseacetate phthalate. Further details on techniques for formulation for andadministration may be found in the latest edition of Remington'sPharmaceutical Sciences (Maack Publishing Co., Easton, Pa.).

The dietary supplement may comprise one or more inert ingredients,especially if it is desirable to limit the number of calories added tothe diet by the dietary supplement. For example, the dietary supplementof the present invention may also contain optional ingredientsincluding, for example, herbs, vitamins, minerals, enhancers, colorants,sweeteners, flavorants, inert ingredients, and the like. For example,the dietary supplement of the present invention may contain one or moreof the following: ascorbates (ascorbic acid, mineral ascorbate salts,rose hips, acerola, and the like), dehydroepiandosterone (DHEA), Fo-Tior Ho Shu Wu (herb common to traditional Asian treatments), Cat's Claw(ancient herbal ingredient), green tea (polyphenols), inositol, kelp,dulse, bioflavinoids, maltodextrin, nettles, niacin, niacinamide,rosemary, selenium, silica (silicon dioxide, silica gel, horsetail,shavegrass, and the like), spirulina, zinc, and the like. Such optionalingredients may be either naturally occurring or concentrated forms.

In some embodiments, the dietary supplements further comprise vitaminsand minerals including, but not limited to, calcium phosphate oracetate, tribasic; potassium phosphate, dibasic; magnesium sulfate oroxide; salt (sodium chloride); potassium chloride or acetate; ascorbicacid; ferric orthophosphate; niacinamide; zinc sulfate or oxide; calciumpantothenate; copper gluconate; riboflavin; beta-carotene; pyridoxinehydrochloride; thiamin mononitrate; folic acid; biotin; chromiumchloride or picolonate; potassium iodide; sodium selenate; sodiummolybdate; phylloquinone; vitamin D3; cyanocobalamin; sodium selenite;copper sulfate; vitamin A; vitamin C; inositol; potassium iodide.Suitable dosages for vitamins and minerals may be obtained, for example,by consulting the U.S. RDA guidelines.

In further embodiments, the compositions comprise at least one foodflavoring such as acetaldehyde (ethanal), acetoin (acetylmethylcarbinol), anethole (parapropenyl anisole), benzaldehyde (benzoicaldehyde), N butyric acid (butanoic acid), d or 1 carvone (carvol),cinnamaldehyde (cinnamic aldehyde), citral (2,6 dimethyloctadien 2,6 al8, gera nial, neral), decanal (N decylaldehyde, capraldehyde, capricaldehyde, caprinaldehyde, aldehyde C 10), ethyl acetate, ethyl butyrate,3 methyl 3 phenyl glycidic acid ethyl ester (ethyl methyl phenylglycidate, strawberry aldehyde, C 16 aldehyde), ethyl vanillin, geraniol(3,7 dimethyl 2,6 and 3,6 octadien 1 ol), geranyl acetate (geraniolacetate), limonene (d, l, and dl), linalool (linalol, 3,7 dimethyl 1,6octadien 3 ol), linalyl acetate (bergamol), methyl anthranilate (methyl2 aminobenzoate), piperonal (3,4 methylenedioxy benzaldehyde,heliotropin), vanillin, alfalfa (Medicago sativa L.), allspice (Pimentaofficinalis), ambrette seed (Hibiscus abelmoschus), angelic (Angelicaarchangelica), Angostura (Galipea officinalis), anise (Pimpinellaanisum), star anise (Illicium verum), balm (Melissa officinalis), basil(Ocimum basilicum), bay (Laurus nobilis), calendula (Calendulaofficinalis), (Anthemis nobilis), capsicum (Capsicum frutescens),caraway (Carum carvi), cardamom (Elettaria cardamomum), cassia,(Cinnamomum cassia), cayenne pepper (Capsicum frutescens), Celery seed(Apium graveolens), chervil (Anthriscus cerefolium), chives (Alliumschoenoprasum), coriander (Coriandrum sativum), cumin (Cuminum cyminum),elder flowers (Sambucus canadensis), fennel (Foeniculum vulgare),fenugreek (Trigonella foenum graecum), ginger (Zingiber officinale),horehound (Marrubium vulgare), horseradish (Armoracia lapathifolia),hyssop (Hyssopus officinalis), lavender (Lavandula officinalis), mace(Myristica fragrans), marjoram (Majorana hortensis), mustard (Brassicanigra, Brassica juncea, Brassica hirta), nutmeg (Myristica fragrans),paprika (Capsicum annuum), black pepper (Piper nigrum), peppermint(Mentha piperita), poppy seed (Papayer somniferum), rosemary (Rosmarinusofficinalis), saffron (Crocus sativus), sage (Salvia officinalis),savory (Satureia hortensis, Satureia montana), sesame (Sesamum indicum),spearmint (Mentha spicata), tarragon (Artemisia dracunculus), thyme(Thymus vulgaris, Thymus serpyllum), turmeric (Curcuma longa), vanilla(Vanilla planifolia), zedoary (Curcuma zedoaria), sucrose, glucose,saccharin, sorbitol, mannitol, aspartame. Other suitable flavoring aredisclosed in such references as Remington's Pharmaceutical Sciences,18th Edition, Mack Publishing, p. 1288-1300 (1990), and Furia andPellanca, Fenaroli's Handbook of Flavor Ingredients, The Chemical RubberCompany, Cleveland, Ohio, (1971), known to those skilled in the art.

In other embodiments, the compositions comprise at least one syntheticor natural food coloring (e.g., annatto extract, astaxanthin, beetpowder, ultramarine blue, canthaxanthin, caramel, carotenal, betacarotene, carmine, toasted cottonseed flour, ferrous gluconate, ferrouslactate, grape color extract, grape skin extract, iron oxide, fruitjuice, vegetable juice, dried algae meal, tagetes meal, carrot oil, cornendosperm oil, paprika, paprika oleoresin, riboflavin, saffron, tumeric,tumeric and oleoresin).

In still further embodiments, the compositions comprise at least onephytonutrient (e.g., soy isoflavonoids, oligomeric proanthcyanidins,indol 3 carbinol, sulforaphone, fibrous ligands, plant phytosterols,ferulic acid, anthocyanocides, triterpenes, omega 3/6 fatty acids,conjugated fatty acids such as conjugated linoleic acid and conjugatedlinolenic acid, polyacetylene, quinones, terpenes, cathechins, gallates,and quercitin). Sources of plant phytonutrients include, but are notlimited to, soy lecithin, soy isoflavones, brown rice germ, royal jelly,bee propolis, acerola berry juice powder, Japanese green tea, grape seedextract, grape skin extract, carrot juice, bilberry, flaxseed meal, beepollen, ginkgo biloba, primrose (evening primrose oil), red clover,burdock root, dandelion, parsley, rose hips, milk thistle, ginger,Siberian ginseng, rosemary, curcumin, garlic, lycopene, grapefruit seedextract, spinach, and broccoli.

In still other embodiments, the compositions comprise at least onevitamin (e.g., vitamin A, thiamin (B1), riboflavin (B2), pyridoxine(B6), cyanocobalamin (B12), biotin, ascorbic acid (vitamin C), retinoicacid (vitamin D), vitamin E, folic acid and other folates, vitamin K,niacin, and pantothenic acid). In some embodiments, the particlescomprise at least one mineral (e.g., sodium, potassium, magnesium,calcium, phosphorus, chlorine, iron, zinc, manganese, flourine, copper,molybdenum, chromium, selenium, and iodine). In some particularlypreferred embodiments, a dosage of a plurality of particles includesvitamins or minerals in the range of the recommended daily allowance(RDA) as specified by the United States Department of Agriculture. Instill other embodiments, the particles comprise an amino acid supplementformula in which at least one amino acid is included (e.g., l-carnitineor tryptophan).

C. Uses of Krill Oil

Previously, it was disclosed that omega-3 fatty acids haveanti-inflammatory properties. See, e.g., Calder. Am. J. Clin. Nutr. 83(2006) 1505S. In addition, in it was disclosed that a phospholipidemulsion derived from a marine and/or synthetic origin comprisingpolyunsaturated fatty acids have anti-inflammatory and/orimmuno-suppressive effects. See, e.g., U.S. Pat. No. 5,434,183. Anembodiment of this invention is a krill oil composition effective forreducing inflammation i.e. reducing the levels of TNF-α, IL-1 beta,IL-6, IL-10, TGF beta and fibrinogen in the blood.

Type 2 diabetes is a metabolic disorder characterized by impairedglycemic control (high blood glucose levels). In type 2 diabetes, it isthe tissue wide insulin resistance that contributes to the developmentof the disease. Strategies reducing insulin resistance or improvingtissue sensitivity to insulin are recognized as beneficial in preventingtype 2 diabetes. In healthy humans, a 3-week supplementation with fishoil (1.1 g EPA/d and 0.7 g DHA/d) decreased the insulin response to anoral glucose load by 40%. Omega-3 PUFA dietary enrichment resulted inlower glucose oxidation, higher fat oxidation, and increased glycogenstorage; the glycemic response was unchanged, however, which indicatesan improved sensitivity to insulin. In another embodiment of thisinvention is a krill oil composition effective for reducing the insulinresistance.

Krill oil has not been disclosed as being effective in treating one ofthe most important life style problems of modern societies, i.e., excessweight gain and obesity. Excess adipose tissue mass (overweight andobesity) is associated with low grade inflammation in adipose tissue andin the whole body reflecting the inflammatory mediators “spilling over”from fat tissue. Trayhurn et al., Br. J. Nutrition (2004), 92(3),347-355. Inflammation appears to be an important link between obesityand metabolic syndrome/type-II diabetes as well as cardiovasculardisease. Libby et al., J. Amer. Coll. Card. (2006), 48(9, Suppl. A),A33-A46. Thus, excess adipose tissue is an unhealthy condition. Weightreduction will improve the inflammatory condition, but persistent weightreduction is difficult to achieve. Omega-3 fatty acid supplementationmay alleviate the inflammatory condition in adipose tissue and thusideally complement the principal strategies of weight reduction i.e. lowcalorie diet and exercise. There are clinical studies in humans thatdemonstrate that omega-3 enhance the effect of very low calorie diet andexercise in reducing body fat mass. Kunesova et al., Physiologicalresearch/Academia Scientiarum Bohemoslovaca (2006), 55(1), 63-72.Although diet and exercise regime may fail to result in consistentdecrease in weight in long term, the effect of omega-3 fatty acidsalleviating the inflammatory condition in the adipose tissue may persistgenerating a condition that can be described as “healthy adiposetissue”. Previously, it was shown that dietary omega-3 fatty acids canbe used to reduce inflammation in adipose tissue without influencinglevel of obesity. Todoric et al., Diabetologia (2006), 49(9), 2109-2119.Reduction in adipose tissue inflammation was demonstrated by an increasein circulating levels of adiponectin. Adiponectin is an adipose tissuederived anti-inflammatory hormone. Results on the treatment of obesepeople with omega-3 fatty acids to alleviate circulating levels ofinflammatory markers are inconclusive. Trebble et al., Br. J. Nutrition(2003), 90(2), 405-412. However, duration of these studies may not havebeen sufficient given the slow turnover of adipose tissue in humans.Itoh et al. found that 1.8 g/d of EPA increased adiponectin, a marker ofadipose tissue derived inflammation, in a group of overweight subjectswith metabolic syndrome. Itoh et al., Arteriosclerosis, Thrombosis, andVascular Biology (2007), 27(9), 1918-1925.

An embodiment of the invention is the use of krill oil to increase serumadiponectin levels. Adiponectin is a protein hormone that modulates anumber of metabolic processes, including glucose regulation and fattyacid catabolism. Adiponectin is exclusively secreted from adipose tissueinto the bloodstream and is very abundant in plasma relative to manyhormones. Levels of the hormone are inversely correlated with body massindex (BMI). The hormone plays a role in alleviating the metabolicdysregulation that may result in type 2 diabetes, obesity,atherosclerosis and non-alcoholic fatty liver disease (NAFLD). Diez etal., Eur. J. Endocrinol. 148 (3): 293-300; Ukkola et al., J. Mol. Med.80 (11): 696-702.

Another embodiment of the invention is to use krill oil in an overweightand obese subjects for alleviating diet induced adipose tissuedysfunction and diet induced changes in the lipid metabolism.

In further embodiments, krill oil is effective in reducing risk factorsof type 2 diabetes such as hyperinsulinemia and insulin resistance andcardiovascular disease risk factors in overweight subjects. In additionthis invention discloses that krill oil is effective in preventingaccumulation of fat in muscles and in the liver (liver steatosis).

It is well known in the art that the obese Zucker rat is a useful ratmodel to study metabolic Syndrome X and non-insulin dependent diabetesmellitus, including glucose tolerance, insulin resistance andhyperinsulinaemia. It has also been shown previously that astaxanthin isa powerful antioxidant, useful for prevention of oxidative stress invivo and in Zucker rats using vitamin E. See, e.g., Aoi et al., (2003).Antioxidants & Redox Signaling. 5(1):139-44; Laight et al., Eur. J.Pharmacol. 377 (1999) 89.

In yet another embodiment of the invention is a krill oil compositioneffective of improving the blood lipid profile by increasing the HDLcholesterol levels, decreasing the LDL cholesterol and triglyceridelevels. Hence the novel krill oil composition is effective for treatingmetabolic syndrome. Metabolic syndrome is defined as the coexistence of3 or more components selected from the group: abdominal obesity, highserum triglyceride levels, low HDL levels, elevated blood pressure andhigh fasting plasma glucose levels.

In another embodiment of the invention, the krill oil compositions arefound to be effective and safe for the treatment of metabolic syndromein humans.

In still other embodiments, the krill oil compositions of the presentinvention find use in increasing or inducing diuresis. In someembodiments, the krill oil compositions of the present invention finduse in decreasing protein catabolism and increasing the muscle mass of asubject.

In some embodiments, the kill oil composition of the present inventionfind use in the treatment of fatty heart disease and non-alcoholic fattyacid liver disease. Thus, the krill oil compositions are useful fordecreasing the lipid content of the heart and/or liver and/or muscle ofa subject.

In yet another embodiment of the invention is a method to increase thetransfer of DHA to the brain.

Example 1

Antarctic krill (Euphausia superba) was captured and brought on boardalive, before it was processed into krill meal, an oil (asta oil) andstickwater. The composition and properties of the krill meal wasmonitored during the processing and compared to a commercial competitor(Table 1 and 2). Furthermore, the amino acid composition of the krillmeal and stickwater was determined (Table 3), showing that krill meal isa suitable feed source for to be used in aquaculture due to thepresences of all the essential amino acids teleost fish require. Duringthe krill meal processing a neutral oil (asta oil) is recovered, thechemical composition of the asta oil is shown in Tables 4 and 5.

TABLE 1 Composition of products from the processing line KonstruktorKoshkin Round frozen After After (Ukranian krill decanter drier vessel)Protein 13.5 g/100 g 20.9 g/100 g 58.5 g/100 g 60.2 g/100 g Moisture76.3 g/100 g 65.6 g/100 g  9.1 g/100 g  9.6 g/100 g Lipid (Folch)  8.6g/100 g   10 g/100 g 21.8 g/100 g 21.4 g/100 g Free fatty 29.8 g/100 g25.3 g/100 g 24.8 g/100 g 23.3 g/100 g acids Total 53.3 mg/kg 81.3 mg/kg145 mg/kg 126 mg/kg astaxanthin

TABLE 2 Lipid class composition in products from the processing lineKonstruktor Round Koshkin frozen After After (Ukranian krill decanterdrier vessel) Crude protein (g/100 g) (g/100 g) (g/100 g) (g/100 g) Waxester/ 2.5 3.0 1.9 3.3 cholesterol ester Triglycerides/ 30.2 33.7 29.332.2 pigments Free fatty acids 15.1 2.5 9.0 5.9 Monoglycerides 3.9 Nd1.3 Nd PE 6.6 10.4 7.9 6.3 PS 1.2 1.6 1.4 2.7 PI 1.9 2.0 2.1 3.5 PC 2835.9 32.0 32.1 Sphingomyeline/ 2.0 0.5 3.0 3.0 lyso PC Nd = not detected

TABLE 3 Amino acids in krill meal and stick water Total in meal Free inmeal Free in stickwater (g/100 g (g/100 g (g/100 g Amino acid protein)protein) protein) Aspartic acid 10.5 0.02 0.22 Glutamic acid 13.5 0.0070.51 Hydroxiproline <0.5 <0.001 <0.05 Serine 4.2 0.02 0.13 Glycine 4.40.18 3.28 Histidine 2.1 <0.01 <0.05 Arginine 6.7 0.56 4.86 Threonine 4.1<0.01 0.22 Alanine 5.4 0.08 0.87 Proline 3.8 0.53 2.32 Tyrosine 4.0 0.010.2 Valine 5.0 0.02 0.13 Methionine 2.9 <0.01 0.12 Isoleucine 5.0 0.020.1 Leucine 7.8 0.14 0.19 Phenylalanine 4.4 0.01 0.1 Lysine 7.8 0.020.27 Cysteine/Cystine 1.4 <0.01 <0.05 Thryptophan 1.1 <0.02 <0.05Creatinine <0.01 <0.05 Asparagine <0.01 0.05 Glutamine <0.01 <0.053-aminopropanoic acid 0.5 8.99 Taurine 0.5 8.52 4-aminobutanoic acid<0.01 <0.05 Citrulline 0.04 0.14 Carnosine <0.01 <0.05 Anserine <0.01<0.05 Ornithine 0.02 1.04 3-aminopropanoic acid is also known asβ-alanine 4-aminobutanoic acid is alos known as γ-aminobutyric acid orGABA

TABLE 4 Composition and quality parameters of asta oil. Moisture 0.14g/100 g  Insoluble impurities 0.02 g/100 g  Unsaponifable matter 1.5g/100 g Nitrogen 0.5 g/100 g Free fatty acids 0.3 g/100 g Peroxide value<2 meq peroxide/kg oil Ansidine value <1 Phosphorous  23 mg/kgPhopspholipids 575 mg/kg Astaxanthin 1245 mg/kg 

TABLE 5 Fatty acid composition of the asta oil Fatty Acid File Asta oilC4:0 0.00 C6:0 0.00 C8:0 0.00 C10:0 0.00 C12:0 0.00 C14:0 17.5 C14:10.00 C15:0 0.00 C16:0 19.3 C16:1 9.7 C18:0 1.2 C18:1 22.6 C18:2N6 1.4C18:3N6 0.1 C18:3N3 0.7 C18:4N3 3.0 C20:0 0.1 C20:1 1.3 C20:2N6 <0.1C20:3N6 0.1 C20:4N6 0.1 C20:3N3 <0.1 C20:4N3 0.2 C20:5N3 (EPA) 5.6 C22:00.1 C22:1 0.3 C22:2N6 0.0 C22:4N6 <0.1 C22:5N6 0.00 C22:5N3 0.2 C22:6N3(DHA) 2.00 C24:1 0.03 Total 88.4 Saturated 38.0 Monounsaturated 33.9Polyunsaturated 16.4 Total 88.4 Omega-3 11.9 Omega-6 1.6

Example 2

The krill meal obtained in example 1 was then ethanol extractedaccording to the method disclosed in JP02215351. The results showed thataround 22% fat from the meal could be extracted, somewhat lower than wasextracted using Folch (25%). Table 6 shows the fatty acid composition ofthe krill meal and the krill oil extracted from the meal using ethanol.Table 7 shows the composition and properties of the krill meal andproducts before and after extraction, whereas table 8 shows the lipidcomposition.

TABLE 6 Fatty acid distribution in krill meal (g/100 g lipid) and theethanol extracted krill oil. Fatty Acid File Krill meal EtOH KO C4:00.00 C6:0 0.00 C8:0 0.00 C10:0 0.00 C12:0 0.00 C14:0 7.8 6.4 C14:1 0.00C15:0 0.00 C16:0 15.8 14.7 C16:1 5.1 4.2 C18:0 0.9 0.7 C18:1 13.4 11.8C18:2N6 1.1 1.2 C18:3N6 0.1 0.1 C18:3N3 0.4 0.4 C18:4N3 1.1 0.1 C20:00.1 0.1 C20:1 0.8 0.6 C20:2N6 <0.1 <0.1 C20:3N6 0.1 <0.1 C20:4N6 0.2 0.2C20:3N3 <0.1 <0.1 C20:4N3 0.2 0.2 C20:5N3 (EPA) 10.5 10.4 C22:0 <0.1<0.1 C22:1 0.5 0.4 C22:2N6 <0.1 <0.1 C22:4N6 <0.1 C22:5N6 0.00 C22:5N30.2 C22:6N3 (DHA) 5.4 4.8 C24:1 0.03 Saturated 24.6 21.9 Monounsaturated19.9 17.0 Polyunsaturated 21.0 19.4 Total 65.5 58.2 Omega-3 18.2 17.0Omega-6 1.3

TABLE 7 Composition and properties of the krill meal and products afterextraction EtOH Krill Delipidated extracted meal krill meal krill oilCrude 586 g/kg 735 g/kg protein Fat (Folch) 250 g/kg 30 g/kg Moisture/71 g/kg 134 g/kg 85 g/kg ethanol Astaxanthin 144 mg/kg 10 mg/kg 117mg/kg esters Diesters 110 mg/kg 8.5 mg/kg 117 mg/kg Monoesters 33 mg/kg1.8 mg/kg  37 mg/kg Biological 854 g/kg protein 870 g/kg proteindigestable protein Flow number 4.8 1.9 NH3 9 mg N/100 g 0  3 mg N/100 gTMA 2 mg N/100 g 0 70 mg N/100 g TMAO 125 mg N/100 g  0 456 mg N/100 g 

TABLE 8 Lipid class distribution Delipidated Krill meal krill meal EtOHextracted KO Cholesterol ester 3.5 TG 32.7 37.4 31.1 FFA 7.8 14.1 16.0Cholesterol 9.1 8.0 12.6 DG 1.1 3.3 MG 3.7 Sphingolipid 2.8 PE 6.5 2.52.7 Cardiolipin 4.2 PI 1.1 11.0 PS 1.4 PC 28.6 20.2 25.3 LPC 2.9 2.6 6.2Total polar lipids 40.6 40.5 36.9 Total neutral lipids 54.2 59.5 63.1

Example 3

The krill meal obtained in example 1 was then subjected to asupercritical fluid extraction method in two stages. During stage 1,12.1% fat (neutral krill oil) was removed using neat CO₂ only at 300bars, 60° C. and for 30 minutes. In stage 2, the pressure was increasedto 400 bar and 20% ethanol was added (v/v) for 90 minutes. This resultedin further extraction of 9% polar fat which hereafter is called polarkrill oil. The total fatty acid composition of the polar krill oil, theneutral krill oil and a commercial product obtained from Neptune Biotech(Laval, Quebec, Canada) are listed in Table 9. In addition the fattyacid composition for the phospholipids (Table 10), the neutral lipids(Table 11), the free fatty acids, diglycerides (Table 12),triglycerides, lyso-phosphatidylcholine (LPC) (Table 13),phosphatidylcholine (PC), phosphatidylethanolamine (PE) (Table 14),phosphatidylinositol (PI) and phosphatidylserine (PS) (Table 15) areshown. Table 16 shows the level of astaxanthin and cholesterol for thedifferent fractions.

TABLE 9 Total fatty acids compositions of the krill oil products (%(w/w)) Total Fatty Acids Fatty Acid Neutral Polar File KO KO NKO C4:00.00 0.00 0.00 C6:0 0.00 0.00 0.00 C8:0 0.00 0.00 0.00 C10:0 0.00 0.000.00 C12:0 0.47 0.04 0.24 C14:0 22.08 3.28 12.48 C14:1 0.33 0.01 0.17C15:0 0.58 0.36 0.52 C16:0 27.03 29.25 23.25 C16:1 0.07 0.01 8.44 C18:01.72 1.03 1.42 C18:1 30.29 13.57 18.92 C18:2N6 2.10 1.96 1.71 C18:3N60.30 0.21 0.00 C18:3N3 0.69 1.02 1.32 C18:4N3 0.05 1.81 3.50 C20:0 0.060.00 0.05 C20:1 1.87 0.80 1.16 C20:2N6 0.05 0.05 0.05 C20:3N6 0.22 0.730.04 C20:4N6 0.00 0.00 0.49 C20:3N3 0.09 0.09 0.06 C20:4N3 0.24 0.510.33 C20:5N3 (EPA) 7.33 29.88 16.27 C22:0 0.01 0.06 0.05 C22:1 0.64 1.780.82 C22:2N6 0.00 0.00 0.00 C22:4N6 0.00 0.00 0.07 C22:5N6 0.00 0.030.00 C22:5N3 0.21 0.67 0.36 C22:6N3 (DHA) 3.51 12.61 8.17 C24:0 0.050.00 0.01 C24:1 0.03 0.25 0.11 Total 100.00 100.00 100.00 Saturated52.00 34.01 38.01 Monounsaturated 33.22 16.43 29.61 Polyunsaturated14.77 49.56 32.37 Total 100.00 100.00 100.00 Omega-3 12.11 46.58 30.02Omega-6 2.67 2.98 2.35

TABLE 10 Fatty acid composition of the phospholipid fraction (% (w/w)).Total Phospholipid Fatty Acid Neutral Polar Neptune File KO KO KO C4:00.00 0.00 0.00 C6:0 0.00 0.00 0.00 C8:0 0.00 0.00 0.00 C10:0 0.00 0.000.00 C12:0 0.00 0.00 0.00 C14:0 0.01 0.00 0.00 C14:1 0.42 0.01 0.01C15:0 2.52 0.00 0.00 C16:0 4.73 35.78 32.81 C16:1 0.19 0.17 0.19 C18:06.31 1.18 1.55 C18:1 38.40 15.58 13.54 C18:2N6 4.18 2.16 1.90 C18:3N60.18 0.22 0.19 C18:3N3 1.02 1.05 1.48 C18:4N3 3.08 1.62 2.15 C20:0 0.270.00 0.07 C20:1 2.55 1.02 0.78 C20:2N6 0.19 0.06 0.06 C20:3N6 0.00 0.140.10 C20:4N6 0.57 0.62 0.64 C20:3N3 0.43 0.08 0.09 C20:4N3 0.17 0.450.42 C20:5N3 (EPA) 20.58 25.53 26.47 C22:0 0.14 0.06 0.00 C22:1 0.002.09 1.94 C22:2N6 0.25 0.71 0.85 C22:4N6 0.44 0.00 0.03 C22:5N6 0.110.00 0.00 C22:5N3 0.00 0.60 0.63 C22:6N3 (DHA) 10.93 10.30 13.34 C24:01.77 0.30 0.37 C24:1 0.59 0.28 0.38 Total 100.00 100.00 100.00 Saturated15.74 37.32 34.81 Monounsaturated 42.14 19.15 16.84 Polyunsaturated42.12 43.53 48.34 Total 100.00 100.00 100.00 Omega-3 36.22 39.62 44.56Omega-6 5.91 3.90 3.78

TABLE 11 Fatty acid composition of the total neutral lipid fraction (%(w/w)). Total neutral lipid Fatty Acid Neutral Polar Neptune File KO KOKO C4:0 0.00 0.00 0.00 C6:0 0.00 0.00 0.00 C8:0 0.00 0.00 0.00 C10:00.00 0.00 0.00 C12:0 0.00 0.00 0.00 C14:0 20.35 11.31 18.44 C14:1 0.300.29 0.25 C15:0 0.53 1.53 0.62 C16:0 23.79 0.49 24.11 C16:1 12.42 5.2211.86 C18:0 1.54 3.27 1.67 C18:1 26.81 33.09 23.82 C18:2N6 1.68 2.371.79 C18:3N6 0.20 0.23 0.25 C18:3N3 0.59 0.62 0.03 C18:4N3 0.03 1.270.05 C20:0 0.07 0.00 0.06 C20:1 1.63 1.41 1.39 C20:2N6 0.04 0.00 0.05C20:3N6 0.18 0.94 0.01 C20:4N6 0.00 0.00 0.00 C20:3N3 0.09 0.00 0.01C20:4N3 0.18 0.41 0.23 C20:5N3 (EPA) 5.88 19.26 9.68 C22:0 0.02 0.000.03 C22:1 0.56 0.60 0.53 C22:2N6 0.00 0.00 0.00 C22:4N6 0.00 0.00 0.04C22:5N6 0.01 0.00 0.00 C22:5N3 0.17 0.27 0.22 C22:6N3 (DHA) 2.74 17.224.64 C24:0 0.15 0.00 0.17 C24:1 0.03 0.21 0.06 Total 100.00 100.00100.00 Saturated 46.45 16.60 45.10 Monounsaturated 41.75 40.82 37.91Polyunsaturated 11.80 42.59 16.99 Total 100.00 100.00 100.00 Omega-39.68 39.05 14.86 Omega-6 2.11 3.54 2.14

TABLE 12 Fatty acid composition of the diglyceride and free fatty acids(% (w/w)). Diglycerides Free fatty acids Fatty Acid Neutral PolarNeptune Neutral Polar Neptune File KO KO KO KO KO KO C4:0 0.00 0.00 0.000.00 0.00 0.00 C6:0 0.00 0.00 0.00 0.00 0.00 0.00 C8:0 0.00 0.00 0.000.00 0.00 0.00 C10:0 0.00 0.00 0.00 0.00 0.00 0.00 C12:0 0.00 0.00 0.000.00 0.00 0.00 C14:0 13.85 14.35 12.22 5.86 7.19 5.45 C14:1 0.18 0.000.17 0.05 0.00 0.08 C15:0 0.49 1.08 0.66 0.46 1.60 0.45 C16:0 23.6835.24 25.81 28.30 29.37 21.12 C16:1 9.49 6.80 0.09 3.27 3.08 4.91 C18:01.56 3.63 1.89 1.13 2.43 0.99 C18:1 23.67 19.85 23.82 14.50 14.77 17.41C18:2N6 1.79 0.21 1.90 1.69 0.97 1.86 C18:3N6 0.17 0.00 0.01 0.14 0.000.22 C18:3N3 0.69 0.00 1.19 0.85 0.00 1.34 C18:4N3 1.92 0.00 2.75 1.300.00 2.72 C20:0 0.00 0.00 0.00 0.00 0.00 0.00 C20:1 1.09 0.00 1.01 0.480.00 0.57 C20:2N6 0.00 0.00 0.00 0.00 0.00 0.00 C20:3N6 0.13 0.00 0.000.08 0.00 0.05 C20:4N6 0.45 0.00 0.64 0.78 0.00 1.43 C20:3N3 0.00 0.000.00 0.00 0.00 0.00 C20:4N3 0.35 0.00 0.43 0.39 0.00 0.43 C20:5N3 14.039.80 18.00 24.33 23.57 25.36 (EPA) C22:0 0.18 0.00 0.10 0.00 0.00 0.05C22:1 0.41 0.00 0.57 0.80 0.69 0.37 C22:2N6 0.28 0.00 0.50 0.46 0.000.54 C22:4N6 0.00 0.00 0.00 0.00 0.00 0.00 C22:5N6 0.00 0.00 0.00 0.000.00 0.00 C22:5N3 0.20 0.00 0.27 0.34 0.00 0.32 C22:6N3 4.74 9.04 7.5314.31 16.33 13.95 (DHA) C24:0 0.64 0.00 0.42 0.49 0.00 0.39 C24:1 0.000.00 0.00 0.00 0.00 0.00 Total 100.00 100.00 100.00 100.00 100.00 100.00Saturated 40.40 54.30 41.10 36.24 40.59 28.45 Mono- 34.84 26.64 25.6619.09 18.54 23.34 unsaturated Poly- 24.77 19.06 33.24 44.67 40.87 48.22unsaturated Total 100.00 100.00 100.00 100.00 100.00 100.00 Omega-321.95 18.85 30.18 41.51 39.90 44.13 Omega-6 2.82 0.21 3.05 3.15 0.974.09

TABLE 13 Fatty acid composition of the triglyceride and lyso-phophatidylcholine fractions (% (w/w)). Triglycerides Lyso PC Fatty AcidNeutral Polar Neptune Neutral Polar Neptune File KO KO KO KO KO KO C4:00.00 0.00 0.00 0.00 0.00 0.00 C6:0 0.00 0.00 0.00 0.00 0.00 0.00 C8:00.00 0.00 0.00 0.00 0.00 0.00 C10:0 0.00 0.00 0.00 0.00 0.00 0.00 C12:00.00 0.00 0.00 0.00 0.00 0.00 C14:0 23.06 26.65 25.13 19.38 4.27 2.87C14:1 0.36 0.93 0.36 0.00 0.08 0.00 C15:0 0.56 2.64 0.78 0.00 0.52 0.45C16:0 23.17 4.93 27.80 41.00 44.14 30.56 C16:1 13.68 11.58 0.04 0.001.84 2.24 C18:0 1.52 3.12 1.99 0.76 1.59 1.32 C18:1 27.83 34.39 27.926.65 14.24 11.29 C18:2N6 1.64 2.05 1.92 0.00 1.75 2.07 C18:3N6 0.20 0.000.30 0.00 0.00 0.06 C18:3N3 0.51 0.00 0.00 7.95 0.67 1.75 C18:4N3 1.990.00 4.83 0.00 1.11 2.46 C20:0 0.06 0.00 0.08 0.00 0.00 0.00 C20:1 1.670.00 1.76 0.00 0.52 0.00 C20:2N6 0.04 0.00 0.05 0.00 0.00 0.00 C20:3N60.05 0.00 0.01 0.00 0.00 0.54 C20:4N6 0.00 0.00 0.00 0.00 0.40 0.00C20:3N3 0.05 0.00 0.07 0.00 0.00 0.00 C20:4N3 0.11 0.00 0.17 0.00 0.310.55 C20:5N3 2.10 7.97 4.44 0.00 18.59 28.48 (EPA) C22:0 0.02 0.00 0.040.00 0.00 0.00 C22:1 0.37 0.00 0.42 0.00 1.46 0.91 C22:2N6 0.00 0.000.00 0.00 0.00 0.00 C22:4N6 0.01 0.00 0.01 0.00 0.00 0.00 C22:5N6 0.000.00 0.01 0.00 0.00 0.00 C22:5N3 0.10 0.00 0.16 0.00 0.41 0.62 C22:6N30.67 3.97 1.42 24.26 7.79 13.82 (DHA) C24:0 0.26 1.78 0.26 0.00 0.320.00 C24:1 0.00 0.00 0.03 0.00 0.00 0.00 Total 100.00 100.00 100.00100.00 100.00 100.00 Saturated 48.64 39.12 56.08 61.14 50.83 35.21 Mono-43.90 46.89 30.52 6.65 18.14 14.44 unsaturated Poly - 7.45 13.99 13.4132.20 31.02 50.35 unsaturated Total 100.00 100.00 100.00 100.00 100.00100.00 Omega-3 5.51 11.94 11.11 32.20 28.87 47.69 Omega-6 1.94 2.05 2.300.00 2.15 2.66

TABLE 14 Fatty acid composition of the phosphatidylcholine and thephosphatidylserine fractions (% (w/w)). PC PS Fatty Acid Neutral PolarNeptune Neutral Polar Neptune File KO KO KO KO KO KO C4:0 0.00 0.00 0.000.00 0.00 0.00 C6:0 0.00 0.00 0.00 0.00 0.00 0.00 C8:0 0.00 0.00 0.000.00 0.00 0.00 C10:0 0.00 0.00 0.00 0.00 0.00 0.00 C12:0 0.00 0.00 0.000.00 0.00 0.00 C14:0 0.75 3.29 2.77 7.60 9.52 2.31 C14:1 2.07 0.04 0.020.00 0.00 0.00 C15:0 1.34 0.00 0.00 3.83 0.00 0.00 C16:0 16.65 31.9229.83 30.44 43.61 19.49 C16:1 0.96 0.01 0.17 9.96 3.47 2.79 C18:0 1.331.06 1.33 2.08 3.34 2.24 C18:1 34.34 13.55 11.16 0.00 7.37 11.87 C18:2N610.55 2.27 1.90 0.00 0.00 0.00 C18:3N6 1.44 0.25 0.20 0.00 0.00 0.00C18:3N3 2.49 1.19 1.54 0.00 0.00 0.00 C18:4N3 2.38 1.92 2.41 0.00 0.000.00 C20:0 2.79 0.03 0.05 0.00 0.00 0.00 C20:1 2.42 0.82 0.74 0.00 0.000.00 C20:2N6 0.56 0.05 0.06 0.00 0.00 0.00 C20:3N6 0.67 0.13 0.09 0.000.00 0.00 C20:4N6 1.85 0.61 0.56 0.00 0.00 0.00 C20:3N3 3.94 0.07 0.060.00 0.00 0.33 C20:4N3 4.32 0.50 0.46 0.00 0.00 0.00 C20:5N3 1.08 29.8530.09 25.84 15.81 16.35 (EPA) C22:0 0.00 0.05 0.02 0.00 0.00 0.00 C22:12.77 0.00 1.87 0.00 0.00 0.00 C22:2N6 0.00 0.81 0.97 0.00 0.00 0.00C22:4N6 0.00 0.01 0.02 0.00 0.00 0.00 C22:5N6 1.49 0.01 0.00 0.00 0.000.00 C22:5N3 1.48 0.67 0.68 0.00 0.00 0.00 C22:6N3 0.00 10.53 12.4920.25 16.89 44.63 (DHA) C24:0 2.34 0.10 0.18 0.00 0.00 0.00 C24:1 0.000.25 0.34 0.00 0.00 0.00 Total 100.00 100.00 100.00 100.00 100.00 100.00Saturated 25.19 36.46 34.18 43.95 56.47 24.04 Mono- 42.56 14.67 14.299.96 10.84 14.65 unsaturated Poly- 32.25 48.87 51.53 46.09 32.69 61.31unsaturated Total 100.00 100.00 100.00 100.00 100.00 100.00 Omega-315.69 44.73 47.73 46.09 32.69 61.31 Omega-6 16.56 4.13 3.81 0.00 0.000.00

TABLE 15 Fatty acid composition of the phosphatidylinositol andphophatidylethanolamine fractions (% (w/w)). PI PE Fatty Acid NeutralPolar Neptune Neutral Polar Neptune File KO KO KO KO KO KO C4:0 0.000.00 0.00 0.00 0.00 0.00 C6:0 0.00 0.00 0.00 0.00 0.00 0.00 C8:0 0.000.00 0.00 0.00 0.00 0.00 C10:0 0.00 0.00 0.00 0.00 0.00 0.00 C12:0 0.000.00 0.00 0.00 0.00 0.00 C14:0 11.15 5.82 5.72 14.42 4.60 0.83 C14:13.03 0.66 0.00 0.00 0.00 0.10 C15:0 5.86 1.95 3.18 0.00 1.30 0.23 C16:037.02 30.66 31.39 35.91 31.21 18.38 C16:1 18.05 2.24 1.16 0.00 1.51 0.75C18:0 6.72 2.83 5.56 12.72 16.70 1.84 C18:1 18.15 24.77 14.23 36.9619.91 18.45 C18:2N6 0.00 2.67 0.00 0.00 2.62 0.85 C18:3N6 0.00 0.00 0.000.00 0.00 0.00 C18:3N3 0.00 0.00 0.00 0.00 0.00 0.33 C18:4N3 0.00 0.000.00 0.00 0.00 0.00 C20:0 0.00 0.00 0.00 0.00 0.00 0.00 C20:1 0.00 0.000.00 0.00 0.00 0.00 C20:2N6 0.00 0.00 0.00 0.00 0.00 0.00 C20:3N6 0.000.00 0.00 0.00 0.00 1.15 C20:4N6 0.00 0.00 0.00 0.00 0.00 0.00 C20:3N30.00 0.00 0.00 0.00 0.00 0.00 C20:4N3 0.00 0.00 0.00 0.00 0.00 0.00C20:5N3 0.00 17.60 20.45 0.00 10.76 21.26 (EPA) C22:0 0.00 0.00 0.000.00 0.00 0.00 C22:1 0.00 0.00 0.00 0.00 0.00 0.00 C22:2N6 0.00 0.000.00 0.00 0.00 0.00 C22:4N6 0.00 0.00 0.00 0.00 0.00 0.00 C22:5N6 0.000.00 0.00 0.00 0.00 0.00 C22:5N3 0.00 0.00 0.00 0.00 0.00 0.67 C22:6N30.00 10.79 18.32 0.00 11.39 35.16 (DHA) C24:0 0.00 0.00 0.00 0.00 0.000.00 C24:1 0.00 0.00 0.00 0.00 0.00 0.00 Total 100.00 100.00 100.00100.00 100.00 100.00 Saturated 60.76 41.26 45.84 63.04 53.81 21.28 Mono-39.24 27.67 15.39 36.96 21.42 19.30 unsaturated Poly- 0.00 31.07 38.770.00 24.77 59.42 unsaturated Total 100.00 100.00 100.00 100.00 100.00100.00 Omega-3 0.00 28.40 38.77 0.00 22.15 57.43 Omega-6 0.00 2.67 0.000.00 2.62 1.99

TABLE 16 Compositional data for the novel krill oil composition obtainedand NKO krill oil. Neptune Ethanol Polar Neutral Compounds KO extractedKO KO KO Astaxanthin esters 472 mg/kg 117 mg/kg 580 mg/kg 98 mg/kgAstaxanthin free  11 mg/kg  <1 mg/kg  <1 mg/kg <1 mg/kg Totalcholesterol 1 g/100 g 12 g/100 g <0.5 g/100 g 5.7 g/100 g

Example 4

Neutral lipids were extracted from krill meal (138 kg) using SFE withneat CO₂ (solvent ratio 25 kg/kg) at 500 bar and 75° C. The neutrallipids were fractionated at 200 bar (75° C.) and at 60 bar (35° C.) atseparator S1 and S2, respectively. The extract obtained in S1 (19.6 kg)were characterized and the results can be found in Tables 17A-C. Theextract in table S2 (0.4 kg) were rich in water and were not furtherused. Next, the polar lipids were extracted using CO₂ at 500 bar, 20%ethanol and at a temperature of 75° C. Using a solvent ratio of 32(kg/kg) and collecting an extract of 18.2 kg using a separator at 60bars and 35° C. The polar lipids were collected and analyzed (Tables18A-C). Next, the polar lipids were mixed in a 50/50 ratio with theneutral lipids collected from S1 before finally the ethanol was removedcarefully by evaporation. The product obtained was red and transparent.If the ethanol is removed before the mixing if the fractions atransparent product is not obtained. The composition of the 50/50 redand transparent product can be found in Tables 19A-C.

TABLE 17A Fatty acid composition of the extract collected in S1 Fattyacid Unit Amount 14:0 g/100 g 18.4 16:0 g/100 g 22.2 18:0 g/100 g 1.516:1 n-7 g/100 g 10.9 18:1 (n-9) + (n-7) + (n-5) g/100 g 25.6 20:1(n-9) + (n-7) g/100 g 1.8 22:1 (n-11) + (n-9) + (n-7) g/100 g 0.5 16:2(n-4) g/100 g 1.3 16:4 (n-1) g/100 g 1.2 18:2 n-6 g/100 g 1.3 18:3 n-3g/100 g 0.8 18:4 n-3 g/100 g 2.9 20:5 n-3 g/100 g 4.1 22:6 n-4 g/100 g1.7

TABLE 17B Lipid class composition of the extract collected in S1 LipidUnit Amount Triacylglycerol g/100 g 84 Diacylglycerol g/100 g 0.7 Freefatty acids g/100 g 1.5 Cholesterol g/100 g 2.7 Cholesterol esters g/100g 0.9

TABLE 17C Miscellaneous analysis of the extract in S1. Compound UnitAmount Free astaxanthin mg/kg 4.3 Astaxanthin esters mg/kg 462Trimethylamin mg N/100 g <1 Trimethylamineoxide mg N/100 g 2

TABLE 18A Fatty acid composition of the extract collected after CO₂ and20% ethanol in S1. Fatty acid Unit Amount 14:0 g/100 g 1.3 16:0 g/100 g13.8 18:0 g/100 g 0.6 16:1 n-7 g/100 g 0.9 18:1 (n-9) + (n-7) + (n-5)g/100 g 6.5 20:1 (n-9) + (n-7) g/100 g 0.6 22:1 (n-11) + (n-9) + (n-7)g/100 g 0.1 16:2 (n-4) g/100 g <0.1 16:4 (n-1) g/100 g <0.1 18:2 n-6g/100 g 0.8 18:3 n-3 g/100 g 0.6 18:4 n-3 g/100 g 1.0 20:5 n-3 g/100 g14.7 22:6 n-4 g/100 g 6.5

TABLE 18B Lipid class composition of the extract collected after CO₂ and20% ethanol in S1. Lipid Unit Amount Triacylglycerol g/100 g <0.5Cholesterol g/100 g <0.5 Phophatidylethanolamine g/100 g 1.6Phosphatidylcholine g/100 g 67 Lyso-phophatidylcholine g/100 g 4.4

TABLE 18C Miscellaneous analysis of the extract in S1. Compound UnitAmount Trimethylamin mg N/100 g 422 Trimethylamineoxide mg N/100 g 239

TABLE 19A Fatty acid composition of the final blended product obtainedin Example 4 in S1. Fatty acid Unit Amount 14:0 g/100 g 9.7 16:0 g/100 g18.5 18:0 g/100 g 1.0 16:1 n-7 g/100 g 5.8 18:1 (n-9) + (n-7) + (n-5)g/100 g 16.0 20:1 (n-9) + (n-7) g/100 g 1.2 22:1 (n-11) + (n-9) + (n-7)g/100 g 1.0 16:2 (n-4) g/100 g 0.3 16:4 (n-1) g/100 g <0.1 18:2 n-6g/100 g 1.0 18:3 n-3 g/100 g 0.8 18:4 n-3 g/100 g 2.1 20:5 n-3 g/100 g10.7 22:6 n-4 g/100 g 4.7

TABLE 19B Lipid class composition of the final blended product obtainedin Example 4. Lipid Unit Amount Triacylglycerol g/100 g 53Diacylglycerol g/100 g 1.3 Free fatty acids g/100 g 0.5 Cholesterolg/100 g 0.6 Cholesterol esters g/100 g <0.5 Phophatidylethanolamineg/100 g <1 Phosphatidylcholine g/100 g 42 Lyso-phophatidylcholine g/100g 5.9

TABLE 19C Miscellaneous analysis of the final blended product obtainedin example 4. Compound Unit Amount Free astaxanthin mg/kg 1.1Astaxanthin esters mg/kg 151 Trimethylamin mg N/100 g 109Trimethylamineoxide mg N/100 g 80

Example 5

The asta oil obtained in example 1 was blended with the polar lipidsobtained in example 4 in a ratio of 46:54 (v/v). Next the ethanol wasremoved by evaporation and a dark red and transparent product wasobtained. The product was analyzed and the results can be found inTables 20A-C. Furthermore, the product was encapsulated into soft gelssuccessfully. During the encapsulation it was observed that any furtherincrease in phospholipids and thereby viscosity will make it verydifficult to encapsulate the final product.

TABLE 20A Fatty acid composition of the final blended product obtainedin Example 5. Fatty acid Unit Amount 14:0 g/100 g 8.2 16:0 g/100 g 17.718:0 g/100 g 1.0 16:1 n-7 g/100 g 4.9 18:1 (n-9) + (n-7) + (n-5) g/100 g14.9 20:1 (n-9) + (n-7) g/100 g 1.1 22:1 (n-11) + (n-9) + (n-7) g/100 g1.0 16:2 (n-4) g/100 g 0.4 16:4 (n-1) g/100 g <0.1 18:2 n-6 g/100 g 1.218:3 n-3 g/100 g 0.8 18:4 n-3 g/100 g 1.8 20:5 n-3 g/100 g 10.6 22:6 n-4g/100 g 4.8

TABLE 20B Lipid class composition of the final blended product obtainedin Example 5. Lipid Unit Amount Triacylglycerol g/100 g 41Diacylglycerol g/100 g 0.8 Free fatty acids g/100 g 1.2 Cholesterolg/100 g 0.4 Cholesterol esters g/100 g 0.3 Phophatidylethanolamine g/100g 0.6 Phosphatidylcholine g/100 g 51 Lyso-phophatidylcholine g/100 g<0.5 Total polar lipids g/100 g 52.4 Total neutral lipids g/100 g 43.6

TABLE 20C Miscellaneous analysis of the final blended product obtainedin Example 5 Compound Unit Amount Free astaxanthin mg/kg 12 Astaxanthinesters mg/kg 1302 Trimethylamin mg N/100 g 193 Trimethylamineoxide mgN/100 g 1.7

Example 6

Fresh krill was pumped from the harvesting trawl directly into anindirect steam cooker, and heated to 90 C. Water and a small amount ofoil were removed in a screw press before ethoxyquin (antioxidant) wasadded and the denatured meal was dried under vacuum at a temperature notexceeding 80 C. After 19 months storage in room temperature, a sample ofthe denatured meal was extracted in two steps with supercritical CO₂ inlaboratory scale at a flow rate of 2 ml/min at 100 C and a pressure of7500 psi. In the second step 20% ethanol was added to the CO₂. The twofractions collected were combined and analyzed by HPLC using ELSdetection. The phosphatidylcholine was measured to 42.22% whereas thepartly decomposed phosphatidylcholine was 1.68%. This data stronglycontrasts the data obtained by analysis of a krill oil sample in themarketplace that showed a content of 9.05% of phosphatidylcholine and4.60% of partly decomposed phosphatidylcholine.

Example 7

Krill lipids were extracted from krill meal (a food grade powder) usingsupercritical fluid extraction with co-solvent. Initially, 300 barpressure, 333° K and 5% ethanol (ethanol: CO₂, w/w) were utilized for 60minutes in order to remove neutral lipids and astaxanthin from the krillmeal. Next, the ethanol content was increased to 23% and the extractionwas maintained for 3 hours and 40 minutes. The extract was thenevaporated using a falling film evaporator and the resulting krill oilwas finally filtered. The product obtained was then analyzed and theresults can be found in Table 21.

TABLE 21 Analysis of the krill oil obtained using supercritical fluidextraction. Parameter Value Ethanol 1.11% w/w Water Content 2.98% w/wC20:5 n-3 (EPA) 19.9 C22:6 n-3 (DHA) 11.3 Total Omega 3 35.7 Total Omega6  3.0 Total Phospholipids 50.55 wt % Ratio Omega3-PL/Total Omega 377.6% w/w Ratio EPA-PL/Total EPA 84.4% w/w Ratio DHA-PL/Total DHA 74.7%w/w Triglycerides 25.9 g/100 g Astaxanthin 2091 mg/kg Peroxide Value<0.1

Example 8

Krill oil was prepared according to the method described in example 7extracting from the same krill meal. The oil was subjected to ³¹P NMRanalysis for the identification and quantification of the various formsof phospholipids. The analysis was performed according to the followingmethods: Samples (20-40 mg) were weighed into 1.5 ml centrifuge tubes.Next, NMR detergent (750 μl-10% Na cholate, 1% EDTA, pH 7.0 in H₂O+D₂O,0.3 g L-1 PMG internal standard) was added. Next, the tube was placed ina oven at 60° C. and periodically shaken/sonicated until completelydispersed. The solution was then transferred to a 5 ml NMR tube foranalysis. Phosphorus NMR spectra were recorded on the two-channel BrukerAvance300 with the following instrument settings: spectrometer frequency121.498 MHz, sweep width 24,271 Hz, 64,000 data points, 30 degreeexcitation pulse, 576 transients were normally taken, each with an 8second delay time and f.i.d. acquisition time of 1.35 sec. Spectra wereprocessed with a standard exponential weighting function with 0.2 Hzline broadening before Fourier transformation.

Peaks were identified using known chemical shifts. Deacylation ofsamples with monomethylamine was also used on two samples forconfirmation of peak identity and to achieve better peak resolution.Example spectra are presented in FIG. 1. Peak area integration gaverelative molar amounts of each lipid class. Weight percent values werecalculated using molecular masses calculated from a krill sample fattyacid profile (average chain length=18.6). Total PL levels werecalculated from the PMG internal standard peak. The quantification ofthe phospholipids are shown in table 25 for both the raw material, thefinal product and for a commercially available krill oil (Neptune KrillOil). The main polar ether lipids of the krill meal arealkylacylphosphatidylcholine (AAPC) at 7-9% of total polar lipids,lyso-alkylacylphosphatidylcholine (LAAPC) at 1% of total polar lipids(TPL) and alkylacylphosphatidyl-ethanolamine (AAPE) at <1% of TPL.

TABLE 22 Phospholipid profiles Krill Oil obtained Type B krill powderNKO in Example 7 PC 66.0 68.6 75.3 AAPC 12.0 7.0 13.0 PI 1LPC 1.2 1.30.4 PS 2LPC 7.4 13.8 2.9 LAAPC 2.2 1.2 0.9 PE 6.0 3.4 3.4 AAPE 1.5 SMGPC 1.3 DHSM NAPE 3.4 CL 5.3 2.1 LPE 0.5 LCL % PL in 8.3 30.0 47.9powder or lipid sampleAnalysis has been carried out on the fatty acid and ether/alcoholprofiles of the AAPC. The following results are presented in Table 23.

TABLE 23 Fatty acid profile of the alkylacylphosphatidylcholine. AAPCalcohol AAPC fatty acid composition composition alcohol % 20:5 (n-3) -46.9%; 16:0 47.6 22:6 (n-3) - 36.1%; 18:1 17.8  18:1 (n-9) - 4.6% 16:114.1  22:5 (n-3) - 2.6% 14:0 10  20:4 (n-6) - 1.9% 18:0 8.6  21:5(n-3) - 1.5% 18:2 5.1  18:2 (n-6) - 0.9% 17:0 4.4  16:1 (n-9) - 0.8%15:0-i 2.1 16:0 - 0.7% 15:0 1.7  phytanic - 0.6% 20:1 1.4  18:3 (n-3) -0.5% 15:0-a 1.3  18:4 (n-3) - 0.4% 18:0-i 0.4  18:1 (n-7) - 0.4% 24:1 -0.4% 14:0 - 0.3%

The rest of alcohols (i17:0, etc.), were less than 0.3% each. Only partof 20:1 was confirmed by GC-MS. Alcohol moieties composition of KrillAAPC was determined (identification was performed in the form of1-alkyl-2,3-diTMS glycerols on GC-MS, % of total fatty alcohols wereobtained by GC with FID). Ten other fatty acids were all below 0.3% bymass.

Example 9

The purpose of this experiment was to investigate the effect ofdifferent omega-3 fatty acid sources on metabolic parameters in theZucker rat. The Zucker rat is a widely used model of obesity and insulinresistance. Obesity is due to a mutation in the leptin receptor whichimpairs the regulation of intake. Omega-3 sources compared in this studywere fish oil (FO) and two types of krill oil. The krill oil were eitherfrom a commercial supplier (Neptune Krill oil) or prepared according toexample 7 (Superba™). Four groups of rats (n=6 per group) were fed adlib either a control diet (CTRL) or a diet supplemented with a source ofomega-3 fatty acids (FO, NKO, Superba). All diets supplied same amountof dietary fatty acids, oleic acid, linoleic acid and linolenic acid.Omega-3 diets (FO, NKO and Superba™) were additionally balanced for EPAand DHA content. The Zucker rats were 4 wk old at the start of the studywith average initial weight of 250 g. At this stage the Zucker rats canbe characterized as being pre-diabetic. Rats were fed the test diets for4 wk after which they were sacrificed and blood and tissue samples werecollected. Data presented in the following figures are means±SE. Thisexample shows that supplementation of the Zucker rat with krill oilprepared as in example 7 results in an improvement of metabolicparameters characteristic of the obesity induced type two diabeticcondition. The effect induced by the novel krill oil is often morepronounced than the effect of FO an in several cases greater than theeffect induced by NKO. Specifically, the effects of the two types ofkrill oil differentiated with respect to the reduction of blood LDLcholesterol levels as well as lipid accumulation in the liver and muscle(FIG. 2-9). Furthermore, the efficacy of transfer of DHA from the dietto the brain tissue was greatest with the krill oil prepared as inexample 7 (FIG. 10).

Example 11

This example describes the effect of the supplementation of human dietswith krill oil, fish oil (positive control), or a negative control oil(no omega-3 fatty acids) on blood urea nitrogen (BUN).

BUN measures the amount of nitrogen in the blood that comes from urea.BUN is used as a measure of renal function. Serum creatinine is,however, considered to be a more specific measure of renal function. Inthis study, krill oil decreased BUN by 11.8% while creatinine levelswere unchanged. Thus, it is likely that the decrease in BUN is due tosome other effect than improved renal function. BUN decreases if krilloil induced diuresis i.e. excretion of urine (diuretic effect).

BUN also decreases if body protein catabolism is reduced. Proteincatabolism is a normal feature of body protein turnover. Many tissuesexpress high protein turnover rates. For example the gastrointestinalsystem expresses high rates of protein turnover. In growing animals areduction in GI protein catabolism improves weight gain. Micesupplemented with krill oil grew at a faster rate than mice supplementedwith fish oil or control diet (FIG. 11).

TABLE 24 The effect on blood urea nitrogen in humans for the differenttreatment groups. Control Krill Oil Menhaden oil n = 23 n = 24 n = 25 pBUN, mg/dL Baseline 11.5 (7.8, 13.8) 11.5 (9.5, 13.5) 11.5 (9.5, 14.0)0.523 Δ from baseline, %  11.0 (−14.3, 26.1) −11.8 (−20.0, 1.5)  9.1(−9.1, 35.7)  0.014r Creatinine, mg/dL Baseline 0.9 (0.7, 0.9) 0.9 (0.7,0.9) 0.8 (0.8, 1.0)   0.952r (r) Δ from baseline, %  0.0 (−9.6, 2.9) 0.0 (−2.0, 5.9)  0.0 (−5.9, 6.7) 0.416

Example 12

The purpose of this experiment was to investigate the effect of dietarykrill oil on metabolic parameters in high-fat fed mice and to comparethe effect of dietary krill oil with that of fish oil containing thesame amount of omega-3 fatty acids. Four groups of C57BL/6 mice (n=10per group) were fed 1) chow (N), 2) high fat diet comprising 21% butterfat and 0.15% cholesterol (HF), 3) high fat diet+krill oil (HFKO) or 4)high fat diet+fish oil (HFFO). Treatment 3 contained 2.25% (w/w) krilloil as prepared in example 5 (except that the astaxanthin content was500 ppm) which were equivalent to 0.36% omega-3 fatty acids. Treatment 4also contained 0.36% omega-3 fatty acids obtained from regular 18-12fish oil. The diets were fed to the mice for 7 weeks with free access todrinking water. Data represented in this example means±SE. Columns notsharing a common letter are significantly different (P<0.05) by ANOVAfollowed by Tukey's multiple comparison test. N=normal chow diet (n=10);HF=high-fat diet (n=10); HFFO=high-fat diet supplemented with fish oil(n=9); HFKO=high-fat diet supplemented with krill oil (n=8). The dataare presented in FIGS. 18-25.

This example shows that supplementation of high-fat fed mice with krilloil results in an amelioration of diet-induced hyperinsulinemia, insulinresistance, increase in muscle lipid content (measured as a change inmuscle mass), serum adiponectin reduction and hepatic steatosis. Thesepotentially beneficial atheroprotective effects were similar or greaterthan those achieved with a supplement containing a comparable level ofomega-3 fatty acids (FIG. 12-19).

1. A method of production of krill oil comprising: a) providing krill;b) treating said krill to denature lipases and phospholipases in saidkrill to provide a denatured krill product; c) extracting oil from saiddenatured krill product with a polar solvent to provide a krill oil withfrom about 3% to about 10% w/w ether phospholipids; from about 27% to50% w/w non-ether phospholipids so that the amount of totalphospholipids in the composition is from about 30% to 60% w/w; and fromabout 20% to 50% w/w triglycerides.
 2. The method of claim 1, whereinsteps a and b are performed on a ship.
 3. The method of claim 1, whereinsaid treating comprises heating.
 4. The method of claim 1, wherein saidkrill is freshly harvested.
 5. The method of claim 1, wherein said krilloil further comprises from about 20% to 35% omega-3 fatty acids as apercentage of total fatty acids in said composition.
 6. The method ofclaim 5, wherein from about 70% to 95% of said omega-3 fatty acids areattached to said phospholipids.
 7. The method of claim 1, wherein saidextracting comprises supercritical fluid extraction with a polarentrainer.
 8. The method of claim 1, further comprising preparing saidkrill oil for encapsulation.
 9. The method of claim 8, furthercomprising encapsulating said krill oil.
 10. The method of claim 1,wherein said krill is Antarctic krill.
 11. The method of claim 10,wherein said Antarctic krill is Euphausia superba.
 12. The method ofclaim 1, wherein said krill is Euphausia pacifica.
 13. A method ofproduction of krill oil comprising: a) providing fresh krill; b)treating said fresh krill to denature lipases and phospholipases in saidfresh krill to provide a denatured krill product; and c) extracting oilfrom said denatured krill product with a polar solvent to provide akrill oil with from about 3% to about 10% w/w ether phospholipids; fromabout 27% to 50% w/w non-ether phospholipids so that the amount of totalphospholipids in the composition is from about 30% to 60% w/w; and fromabout 20% to 50% w/w triglycerides.
 14. The method of claim 13, whereinsteps a and b are performed on a ship.
 15. The method of claim 13,wherein said treating comprises heating.
 16. The method of claim 13,wherein said krill oil further comprises from about 20% to 35% omega-3fatty acids as a percentage of total fatty acids in said composition.17. The method of claim 16, wherein from about 70% to 95% of saidomega-3 fatty acids are attached to said phospholipids.
 18. The methodof claim 13, wherein said extracting comprises supercritical fluidextraction with a polar entrainer.
 19. The method of claim 13, furthercomprising preparing said krill oil for encapsulation.
 20. The method ofclaim 19, further comprising encapsulating said krill oil.
 21. Themethod of claim 13, wherein said krill is Antarctic krill.
 22. Themethod of claim 10, wherein said Antarctic krill is Euphausia superba.23. The method of claim 13, wherein said krill is Euphausia pacifica.24. A method of production of krill oil comprising: a) providing freshkrill; b) treating said fresh krill to denature lipases andphospholipases in said fresh krill to provide a denatured krill product;c) extracting oil from said denatured krill product with a polar solventto provide a krill oil with from about 3% to about 10% w/w etherphospholipids; from about 27% to 50% w/w non-ether phospholipids so thatthe amount of total phospholipids in the composition is from about 30%to 60% w/w; and from about 20% to 50% w/w triglycerides; and d)preparing said krill oil for encapsulation; wherein steps a and b areperformed on a ship.
 25. The method of claim 24, wherein said treatingcomprises heating.
 26. The method of claim 24, wherein said krill oilfurther comprises from about 20% to 35% omega-3 fatty acids as apercentage of total fatty acids in said composition.
 27. The method ofclaim 26, wherein from about 70% to 95% of said omega-3 fatty acids areattached to said phospholipids.
 28. The method of claim 24, wherein saidextracting comprises supercritical fluid extraction with a polarentrainer.
 29. The method of claim 24, further comprising encapsulatingsaid krill oil.
 30. The method of claim 24, wherein said krill isAntarctic krill.